Comparison of rat hepatocyte and differentiated hepatoma cell line cultures as bio indicators of CYP 1A1 inducers in urban air

Cytochrome P450 1A1 CYP1A1 enzymatic activity was evaluated in cultured liver cells, and taken as a biological indicator of the presence of inducers of this isoform in urban airborne particulate matter fraction samples. It is known that CYP1A1 inducers can play an important role in the risk of mutagenesis and carcinogenesis by environmental pollution. A romatic polycyclic hydrocarbons PAH from urban air were collected in the city of Genoa Italy at two sites on two different days of the year. The objective of the study was to compare the inducibility of cultured rat hepatocytes with that of MH1C1 and FaO rat hepatoma cell lines after exposure to a PAH mixture and to a standard compound, such as benzo b fluoranthene B b F . Cytotoxic effects of the tested concentrations were evaluated by means of 3 4,5, dimenthylthyazol 2 yl 2,5 diphenyltetrazolium bromide MTT and lactate dehydrogenase release LDH tests, the potency of inducers by ethoxyresorufin O deethylase EROD assay. The results were in agreement in the three cellular systems: after exposure to the PAH mixture, an induction at low concentrations was observed; whereas no induction, but rather a decrease in activity was shown at higher concentrations; instead, the exposure to pure B b F showed a dose-response relationship in all cells, even at the highest doses. Such a difference between the toxicity of the complex mixture and that of the pure compound could be ascribed to the presence of drug metabolism inhibitors in the mixture, or to interactions between the original components and their metabolites. The finding that the cell lines responded to the CYP1A1 induction in a very efficient way gives further proof of the applicability of this system to environmental biomonitoring.     

CYTOCHROME P450 1A1 EXPRESSION IN CETACEAN INTEGUMENT: IMPLICATIONS FOR DETECTING CONTAMINANT EXPOSURE AND EFFECTS

Contaminant related health risks to marine mammals are typically inferred from the levels of contaminants measured in blubber. Such measurements alone are insufficient to indicate the likelihood of biological effects from contaminant exposure, especially for contaminants that do not bioaccumulate. Cytochrome P450 1A1 (CYP1A1) in mammals is induced by, and involved in, the metabolism of planar halogenated aromatic hydrocarbons and polycyclic aromatic hydrocarbons, chemicals of concern in aquatic systems. CYP1A induction is a molecular response to exposure to these inducers in many vertebrates. Using immunohistochemistry, we semiquantitatively measured CYP1A1 expression in integument (epidermis and blubber) collected by biopsy or at necropsy from 17 species of cetaceans. CYP1A1 expression was detected in all species and, in some cases, varied both within and between species. CYP1A1 expression in mysticetes was comparable to that in odontocetes. Assessing how the differences in contaminant burdens, life history parameters, and physiological condition between individuals, populations, or species affect CYP1A1 expression in cetacean integument is essential to the interpretation of this induction as a biomarker of exposure to and effects of contaminants. Detection of CYP1A1 expression in integument samples offers a relatively simple, non-lethal technique to study biological changes associated with contaminant exposure in cetaceans.     

Potency of various polycyclic aromatic hydrocarbons as inducers of CYP1A1 in rat hepatocyte cultures

A number of highly toxic environmental pollutants including certain polychlorinated dibenzo-p-dioxins (PCDD), polychlorinated dibenzofurans (PCDF), and 'dioxin-like' polychlorinated biphenyls (PCB) are among the most potent agonists of the aryl hydrocarbon receptor (AHR). Induction of cytochrome P4501A1 (CYP1A1) in mammalian cell culture is widely used as a functional parameter for AHR activation providing an estimate for 'dioxin-like' inducing equivalents in extracts from environmental samples. Since a number of polycyclic aromatic hydrocarbons (PAHs) also act as AHR-agonists, the CYP1A1-inducing potencies, measured as induction of 7-ethoxyresorufin O-deethylase (EROD) activity in rat hepatocyte cultures were analyzed for 16 PAHs frequently present in environmental samples. Among these, seven PAHs including benzo[a]pyrene were relatively potent inducers allowing the determination of Induction Equivalency Factors (IEF). For three PAHs including benzo[k]fluoranthene which acted as weak inducers, IEFs were estimated, while six PAHs including acenaphthylene were classified as inactive. Based on different efficacies the concentration-response characteristics of CYP1A1 induction were analyzed in more detail for benzo[a]pyrene, benzo[k]fluoranthene, and acenaphthylene. Benzo[k]fluoranthene was markedly less effective than benzo[a]pyrene as inducer of EROD activity but even more effective than benzo[a]pyrene as inducer of CYP1A1 protein and mRNA. Acenaphthylene was highly more effective on the level of mRNA than on the levels of protein or EROD activity. Further analysis revealed that the low efficacy of acenaphthylene as inducer of CYP1A1 protein and EROD activity is due to its marked cytotoxicity while no clear-cut explanation was found for the differences in efficacy between benzo[k]fluoranthene and benzo[a]pyrene. The EROD-inducing potency of a mixture of 16 PAH was about 2-fold higher than that calculated on the basis of IEFs of the individual constituents of the mixture.     

Induction of cytochrome P450 1A1 expression in captive river otters fed Prudhoe Bay crude oil: evaluation by immunohistochemistry and quantitative RT-PCR

Numerous studies have explored the relationships between exposure to a variety of environmental contaminants, such as polycyclic aromatic hydrocarbons, and induction of cytochrome P450 1A (CYP1A) in different vertebrates. Few controlled studies, however, simulated chronic long-term exposure with repeated non-lethal sampling of the same individuals, which should better represent repeated exposure incidents in animals inhabiting polluted areas. In this study, we investigated the effects of chronic exposure to crude oil on levels of CYP1A1 in endothelial cells of skin biopsies and peripheral mononuclear blood cells in captive river otters (Lontracanadensis) using repeated sampling of the same individuals. We hypothesized that ingestion of oil would result in an increase in levels of CYP1A1 in both targets, and predicted that the relationship between prolonged exposure and expression of CYP1A1 would reach a plateau indicative of continuous detoxification of hydrocarbons. Fifteen wild-caught male otters were acclimated to captivity, and then fed diets containing no oil (control) or diets containing weathered crude oil at 5 mg day^-1 kg^-1 body weight (low-dose) and 50 mg day^-1 kg^-1 body weight (high-dose), at the Alaska Sealife Center in Seward, Alaska, USA. Expression of CYP1A1 was assessed with immunohistochemical analysis of CYP1A1 protein in skin biopsies and by quantitative RT-PCR analysis of CYP1A1 mRNA in mononuclear blood cells. Both assays revealed a decrease between capture and the transfer to captivity, indicating that the enclosure at the Alaska Sealife Center, and the food we offered to the otters were free of potential inducers of CYP1A1. During the exposure period, increases in CYP1A1 expression were registered by both techniques, followed by a decline in CYP1A1 after oil administration ended. Levels of endothelial CYP1A1 in the high-dose group were comparable to those recorded for wild river otters in PWS in 1996 and 1997. Levels of CPY1A1 mRNA in mononuclear blood cells, however, were well below levels recorded for river otters in Prince William Sound, and no correlation was detected between values obtained from the two methods. Thus, our results from this longitudinal study with repeated sampling of the same individuals provide support for the use of cytochrome P450 1A1 as a biomarker for hydrocarbon exposure. Nonetheless, our results also suggest that the induction process of CYP1A1 may be complicated and interacting with other processes in vivo. Such interactions may obscure our ability to describe specific, quantitative, predictable, dose-response relationships between exposure to hydrocarbons and induction of CYP1A1, which are required of reliable biomarkers. Evaluations of such interactions based on theoretical physiological models in live-animals merit further investigation.     

Cryopreserved human hepatocytes as alternative in vitro model for cytochrome P450 induction studies

Induction of cytochrome P450 (CYP) by drugs is one of major concerns for drug-drug interactions. Thus, the assessment of CYP induction by novel compounds is a vital component in the drug discovery and development processes. Primary human hepatocytes are the preferred in vitro model for predicting CYP induction in vivo. However, their use is hampered by the erratic supply of human tissue and donor-to-donor variability. Although cryopreserved hepatocytes have been recommended for short-term applications in suspension, their use in studies on induction of enzyme activity has been limited because of poor attachment and response to enzyme inducers. In this study, we report culture conditions that allowed the attachment of cryopreserved human hepatocytes and responsiveness to CYP inducers. We evaluated the inducibility of CYP1A1/2 and CYP3A4 enzymes in cryopreserved hepatocytes from three human donors. Cryopreserved human hepatocytes were cultured in serum-free medium for 4 d. They exhibited normal morphology and measurable viability as evaluated by the reduction of tetrazolium salts (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) by cellular dehydrogenases. Treatment with beta-naphthoflavone (10 microM) for 3 d increased ethoxyresorufin-O-deethylase activity (CYP1A1/2) by 6- to 11-fold over untreated cultures and increased CYP1A2 messenger ribonucleic acid (mRNA) expression by three- to eightfold. Similarly, treatment of cryopreserved human hepatocytes with rifampicin (25 microM) for 3 d increased testosterone 6 beta-hydroxylase activity (CYP3A4) by five- to eightfold over untreated cultures and increased CYP3A4 mRNA expression by four- to eightfold. The results suggest that cryopreserved human hepatocytes can be a suitable in vitro model for evaluating xenobiotics as inducers of CYP1A1/2 and CYP3A4 enzymes.     

Characterization of human CYP1A1/1A2 induction by DNA microarray and alpha-naphthoflavone

DNA microarrays and real time PCR were used to analyze the mechanism of gene induction by CYP1A1 inducers, beta-naphthoflavone, and omeprazole, in the human hepatocellular carcinoma HepG2 cells. Reproducible and significant inductions were observed in a limited number of genes including CYP1A1 and CYP1A2. Genes induced by omeprazole included several protein tyrosine kinase targets. This result confirmed that omeprazole could modulate gene expressions through protein tyrosine kinase-mediated pathway. Induction ratios were considerably different from CYP1A1 and CYP1A2 (>10-fold) to other induced genes (<5-fold). alpha-Naphthoflavone, which is known as an antagonist to 2,3,7,8-tetrachlorodibenzo-p-dioxin, inhibited the inductions of heme oxygenase 1, glutamate-cysteine ligase (modifier unit), and thioredoxin reductase by beta-naphthoflavone but not those of CYP1A1 and CYP1A2. It unexpectedly enhanced the beta-naphthoflavone-mediated CYP1A1 and CYP1A2 induction. These results suggest that the CYP1A1 and CYP1A2 genes, which share their 5(') enhancer regions, are regulated differently from the other genes.     

The role of protein tyrosine kinases in CYP1A1 induction by omeprazole and thiabendazole in rat hepatocytes

Benzimidazoles compounds like omeprazole (OME) and thiabendazole (TBZ) mediate CYP1A1 induction differently from classical aryl hydrocarbon receptor (AhR) ligands, 3-methylcholanthrene (3-MC) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). To clarify the involvement of an intracellular signal pathway in CYP1A1 induction by OME and TBZ, the TBZ, OME and 3-MC signal-transducing pathways were compared by using specific protein tyrosine kinase inhibitors in primary culture of rat hepatocytes. The effect of OME and TBZ (75-250 microM) on cytochrome P450 1A1 (CYP1A1) expression was therefore studied in primary cultures of rat hepatocytes after 24 h, 48 h and 72 h of exposure. Both compounds provoked a dose- and time-dependent increase in CYP1A1 (EROD activity, protein and mRNA levels), but OME was less effective at all the concentrations and times tested. The mechanism of benzimidazole-mediated induction of CYP1A1 was investigated by comparison with 3-MC, a prototypical AhR ligand. As expected, OME and TBZ were unable to displace [(3)H]-TCDD from its binding sites to the AhR in competitive binding studies. Moreover, classic tyrosine kinase inhibitor herbimycin A (HA) inhibited the two benzimidazoles-mediated CYP1A1 inductions, but only partially inhibited the 3-MC-mediated one. Another two tyrosine kinase inhibitors, Lavendustin A (LA) and genistein (GEN), had no effect on CYP1A1 induction by benzimidazoles and 3-MC. These results are consistent with the implication of a tyrosine kinase, most probably the Src tyrosine kinase, in the mechanism of CYP1A1 induction in rat hepatocytes.     

Effects of Oral Anorexiant Sibutramine on the Expression of Cytochromes P450s in Human Hepatocytes and Cancer Cell Lines

Sibutramine is a serotonin–norepine‐phrine reuptake inhibitor that was used for weight‐loss management in obese patients. Even though it was officially withdrawn from the market in 2010, it is still present in some tainted weight‐loss pills (as reported by US Food and Drug Administration). Thus, it is still reasonable to study the effects of this compound. The aim of this work was to investigate the potential of sibutramine to induce CYP1A1/CY3A4 in human cancer cell lines and CYP1A1/2, CYP2A6, CYP2B6, and CYP3A4 in human hepatocytes, a competent model of metabolically active cells. The levels of mRNA and protein of CYP1A1/1A2/3A4/2A6/2B6 were compared with the typical inducers, 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) and rifampicin (RIF) for CYP1A1/2 and for other CYPs, respectively. The mRNA and protein levels of all genes in either cancer cell lines or human hepatocytes were induced when treated with typical inducers but not with sibutramine.     

Regulation of cytochrome P450 2e1 expression by ethanol: role of oxidative stress-mediated pkc/jnk/sp1 pathway

CYP2E1 metabolizes ethanol leading to production of reactive oxygen species (ROS) and acetaldehyde, which are known to cause not only liver damage but also toxicity to other organs. However, the signaling pathways involved in CYP2E1 regulation by ethanol are not clear, especially in extra-hepatic cells. This study was designed to examine the role of CYP2E1 in ethanol-mediated oxidative stress and cytotoxicity, as well as signaling pathways by which ethanol regulates CYP2E1 in extra-hepatic cells. In this study, we used astrocytic and monocytic cell lines, because they are important cells in central nervous system . Our results showed that 100 mM ethanol significantly induced oxidative stress, apoptosis, and cell death at 24 h in the SVGA astrocytic cell line, which was rescued by a CYP2E1 selective inhibitor, diallyl sulfide (DAS), CYP2E1 siRNA, and antioxidants (vitamins C and E). Further, we showed that DAS and vitamin C abrogated ethanol-mediated (50 mℳ) induction of CYP2E1 at 6 h, as well as production of ROS at 2 h, suggesting the role of oxidative stress in ethanol-mediated induction of CYP2E1. We then investigated the role of the protein kinase C/c-Jun N-terminal kinase/specificity protein1 (PKC/JNK/SP1) pathway in oxidative stress-mediated CYP2E1 induction. Our results showed that staurosporine, a non-specific inhibitor of PKC, as well as specific PKCζ inhibitor and PKCζ siRNA, abolished ethanol-induced CYP2E1 expression. In addition, inhibitors of JNK (SP600125) and SP1 (mithramycin A) completely abrogated induction of CYP2E1 by ethanol in SVGA astrocytes. Subsequently, we showed that CYP2E1 is also responsible for ethanol-mediated oxidative stress and apoptotic cell death in U937 monocytic cell lines. Finally, our results showed that PKC/JNK/SP1 pathway is also involved in regulation of CYP2E1 in U937 cells. This study has clinical implications with respect to alcohol-associated neuroinflammatory toxicity among alcohol users.     

Binding of polycyclic aromatic hydrocarbons (PAHs) to teleost aryl hydrocarbon receptors (AHRs)

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous, environmental contaminants that pose a potential risk to fish populations. Both field and laboratory studies suggest that exposure of the early life stages of fish to PAH can mimic the embryotoxic effects of the planar halogenated hydrocarbons (PHHs), the most potent of which is 2,3,7,8-tetrachlorodibenzo-p-dioxin. PHH toxicity is mediated by the aryl hydrocarbon receptor (AHR) and PHH potency is predicted by its AHR-binding affinity and CYP1A induction potency. However, the role of the AHR, if any, in mediating the developmental effects of PAH to fish remains unknown. In this study we looked at the AHR binding affinity of a test set of PAH that had been previously ranked for their potency for inducing teleost CYP1A. PAH that induced CYP1A inhibited [3H]TCDD binding to in vitro-expressed AHRs from rainbow trout and the AHR expressed in PLHC-1 fish hepatoma cells. Generally, the relative rank order for AHR binding affinity predicted the rank order of these same PAH for inducing CYP1A reported in other studies. There was a strong, positive relationship between binding to the PLHC-1 AHR (stimulus) and the EC50s for CYP1A induction (response) in whole juvenile trout and in RTL-W1 cells, but EC50s were much higher than expected for a 1:1 stimulus/response relationship. These data show that the ability of PAH to bind to teleost AHR predicts PAH potency for CYP1A induction. If PAH toxicity is receptor-mediated and predicted by induction potencies, we will have a powerful mechanistic-based tool for rapidly assessing the risk of toxicity to fish of PAH from any source.     

Induction of Two Forms of Eel Cytochrome P450 1A Genes by 3-Methylcholanthrene

In eel (Anguilla japonica), exposure to polyaromatic hydrocarbons such as 3-methylcholanthrene leads to induction of two CYP1A enzymes, CYP1A1 and CYP1A6. We studied the time course and tissue specificity of induction of messenger RNAs for CYP1A1 and CYP1A6 in eel by administering 3-methylcholanthrene intraperitoneally. In both cases, the drug induced a rapid increase of mRNAs and biphasic expression. In the liver, mRNA levels of CYP1A1 and CYP1A6 increased 22-fold at 3 hours and 27-fold at 6 hours after the administration, respectively, showing initial peaks in the induction. After the initial inductions, mRNA levels decreased unexpectedly. Following these temporary decreases, the mRNA levels again increased and reached levels that were 35 and 41 times the basal levels at 24 hours after administration, respectively. CYP1A1 and CYP1A6 resembled each other also in the tissue specificity of gene expression; the expression levels were liver ≫ gill > intestine > kidney. The rapid induction, the biphasic expression, and the tissue-specific expression were common features of gene expression in CYP1A1 and CYP1A6 and may come from common structures of the regulatory regions of the two genes. Received December 7, 1998; accepted February 15, 1999