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Cytochrome P450 1A isoenzymes in brain cells: Expression and inducibility in cultured rat brain neuronal and glial cells
Life Sciences 79 (2006) 2387 – 2394 www.elsevier.com/locate/lifescie
Cytochrome P450 1A isoenzymes in brain cells: Expression and inducibility in cultured rat brain neuronal and glial cells Nidhi Kapoor a , Aditya B. Pant b , Alok Dhawan b , Uppendra N. Dwievedi a , Prahlad K. Seth b,1 , Devendra Parmar b,⁎ b
a Biochemistry Department, Lucknow University, University Road, Lucknow, India Developmental Toxicology Division, Industrial Toxicology Research Centre, P.O. Box 80, Mahatma Gandhi Marg, Lucknow-226 001, India
Received 15 June 2006; accepted 1 August 2006
Abstract Studies initiated to determine the expression of CYP1A1/1A2 isoenzymes in the primary cultures of rat brain neuronal and glial cells revealed significant activity of CYP1A-dependent 7-ethoxyresorufin-o-dealkylase (EROD) in microsomes prepared from both rat brain neuronal and glial cells. RT-PCR and immunocytochemical studies demonstrated constitutive mRNA and protein expression of CYP1A1 and 1A2 isoenzymes in cultured neuronal and glial cells. Cultured neurons exhibited relatively higher constitutive mRNA and protein expression of CYP1A1 and 1A2 isoenzymes, associated with higher activity of EROD than the glial cells. Induction studies with 3-methylchlorantherene (MC), a known CYP1Ainducer, resulted in significant concentration dependent increase in the activity of EROD in cultured rat brain cells with glial cells exhibiting a greater magnitude of induction than the neuronal cells. This difference in the increase in enzyme activity was also observed with RT-PCR and immunocytochemical studies, indicating relatively higher increase in CYP1A1 and 1A2 mRNA as well as protein expression in the cultured glial cells when compared to the neuronal cells. The greater magnitude of induction of CYP1A1 in glial cells is of significance, as these cells are components of the blood–brain barrier and it is suggested that they have a potential role in the toxication–detoxication mechanism. Our data indicating differences in the expression and sensitivity of CYP1A1 isoenzymes in cultured rat brain cells will not only help in identifying and distinguishing xenobiotic metabolizing capability of these cells but also in understanding the vulnerability of these specific cell types towards neurotoxicants. © 2006 Elsevier Inc. All rights reserved. Keywords: Cytochrome P450 1A; Neurons; Glia; Induction; mRNA; Enzyme
Introduction Cytochrome P450 (CYP) is a heme containing superfamily of enzymes that metabolize a broad spectrum of endogenous and exogenous compounds (Nelson et al., 1996). CYP1A (CYP1A1 and 1A2) isoenzymes exhibit tissue specificity in constitutive as well as inducible expression. CYP1A1, considered to be predominantly constitutively expressed in extrahepatic tissues, is inducible in liver and extrahepatic tissues while ⁎ Corresponding author. Tel.: +91 522 2613786/2627586x261/262; fax: +91 522 2628227, 2611547. E-mail address: [email protected] (D. Parmar). 1 Present Address: Biotech Park, Sector-G, Jankipuram, Kursi Road, Lucknow, India. 0024-3205/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2006.08.002
CYP1A2, generally known to be a predominantly hepatic form, exhibits both constitutive and inducible expression (Eaton et al., 1995; Guengerich, 1990). CYP1A1 and 1A2 are of toxicological importance because of their role in the bioactivation of environmental toxins and carcinogens such as aromatic amines and polycyclic aromatic hydrocarbons (PAH; Gonzalez et al., 1989; Nebert and Jones, 1989; Omiecinski et al., 1990; Cherng et al., 2001). RT-PCR and immunoblotting studies have revealed the constitutive presence of CYP1A1 and CYP1A2 in rat brain (Kohler et al., 1988; Warner et al., 1988; Anandatheerthavarada et al., 1990; Hodgson et al., 1993; Schilter and Omiecinski, 1993). The marked structural complexity and cellular heterogeneity of brain was reflected in the regional and cellular differences observed in the distribution of CYP1A1/1A2 (Kapitulnik et al.,
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1987; Kohler et al., 1988). Spatial distribution of CYP1A1/1A2 in the different cell types of the brain has indicated endogenous functions. Although the endogenous substrates of CYP1A1/ 1A2 are yet to be identified, the activity of CYP1A isoenzymes is known to be modulated by several neurotransmitters (Agundez et al., 1998). Furthermore, endogenous processes such as a localized inflammatory response in the brain can also modulate its levels, thereby influencing drug response. For tissues exhibiting low regenerative capacity, such as the brain, such modulation would be of major toxicological significance. Although CYPs in brain occur at only 1–5% of the levels of the liver enzyme, it has been reported that the levels of CYPs in specific neurons can be as high or even higher than the levels in hepatocytes (Miksys et al., 2000; Strobel et al., 2001; Parmar et al., 2003; Gervasini et al., 2004). In an attempt to further investigate the distribution of CYPs within the brain, the present study aimed to determine the expression and catalytic activity of CYP1A1 and 1A2 isoenzymes in primary cultured rat brain neuronal (N) and glial cells (G), the two major cell types of the brain. Furthermore, the responsiveness of these cultured rat brain neuronal and glial cells to 3-methylchlorantherene (MC), a CYP1A1/1A2 inducer, was also studied to ascertain the suitability of using these cultured brain cells as a tool for understanding the mechanisms of neurotoxicity and neurodegeneration.
neurons were attached to the PLL coated surface within about 5 min. Unattached cells were then transferred to second and third set of PLL coated flask after which the unattached glial cells were transferred to a fresh PLL coated flask. Only the first and the last batches of flasks were used as a source of N and G cells respectively. Assessment of purity of neuronal and glial cells by immunocytochemistry The purity of neuronal and glial cultures was assessed as described by Kapoor et al. (2006). In brief, the cultured cells were fixed, permeabilized and then incubated with primary antibody (anti-β-III tubulin or anti-GFAP). The cells were then washed and incubated with anti-rabbit FITC labeled secondary antibody, which recognize GFAP, for 1 h. The cells were then washed again and incubated with secondary anti-mouse TRITC labeled antibody, which recognize β-III tubulin. The cells were then mounted with the mounting media and visualized and analyzed under fluorescence microscope (Leica, Germany) using Leica Q Fluoro Software, respectively. The purity of N and G cells preparation, identified by counting multiple fields after staining with cell specific markers, was 90–95%. Treatment of brain cells and cytotoxicity assessment
Materials and methods Chemicals Monoclonal antibodies for GFAP (Chemicon International) and β-III tubulin (Concave company, California) and Secondary Fluorescence antibodies FITC and Rhodamine (TRITC), (Jackson Imunoresearch laboratories, Inc.) were kindly gifted by Dr. Eugene Major, National Institute of Neurological Disorders and Stroke (NINDS), Bethesda, Maryland, U.S.A. Antirat hepatic CYPlAl/1A2 was obtained from Chemicon, USA. All the other chemicals including PCR enzymes and culture reagents were procured from M/s Sigma-Aldrich, St. Louis, MI, U.S.A or Life Technologies, USA. Neuronal and glial cell culture Pregnant albino Wistar rats weighing 175–200 gm (∼ 8 week old) were obtained from the Industrial Toxicology Research Centre breeding colony and raised on a commercial pellet diet and water ad libitum. Animals were cared for in accordance to the policy laid down by the Animal Care Committee of Industrial Toxicology Research Centre and the Ethical Committee of the Centre approved Animal Experimentation. For neuronal cell culture, 14 days old embryos and for glial cells, 0–1 day old pups were used as described in our earlier study by Kapoor et al. (2006). In brief, brain tissue was minced, trypsinised and incubated with DMEM media containing 12% FBS. Cells were then dissociated and the resulting cell suspension were filtered through a 50 μm diameter nylon mesh and pelleted by centrifugation. Approximately, 30 million cells in 12–15 ml medium were plated in each PLL coated flask. More than 90% of the
The cultured cells were treated with different concentrations (0.5-, 1.0-, 2.0-, 3.0-, 4.0- and 8.0 μM) of 3-methychlorantherene (3-MC) for different time intervals (12-, 24-, 48-, 72- and 96 h). Cytotoxicity was assessed by a colorimetric assay based on the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) dye as described by Kapoor et al. (2006). In brief, after the exposure of 3-MC, MTT was added to each well and left for 4-6 h. The medium containing the MTT was aspirated carefully and 150 μl of DMSO was added to each well and incubated for 30 min. The solution in each well was mixed well and the absorbance read on a microplate reader at 550 nm. Enzyme activity The neuronal and glial cells, after treatment with 3-MC were processed for isolation of microsomes as described in our earlier study (Kapoor et al., 2006). The activity of 7-ethoxyresorufinO-deethylase (EROD) was determined in cultured rat brain neuronal and glial cell microsomes by the method of Parmar et al. (1998) using a Perkin Elmer LS 55 Luminescence spectrometer. As isoforms other than CYP1A1 may constitute CYPs in control brain (Warner et al., 1988; Parmar et al., 2003), antibody inhibition studies with anti-CYP1A1/1A2 were also carried out to further identify the role of CYP1A1/1A2 in catalyzing the activity of EROD in brain cells as described earlier (Parmar et al., 1998). Immunocytochemistry For immunocytochemistry, the cultured neuronal and glial cells were fixed, permeabilized and incubated with primary
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antibody for anti-β-III tubulin, anti-CYP1A1/1A2, or antiGFAP and anti-CYP1A1/1A2 as described by Kapoor et al. (2006). The cells were then washed and incubated with antirabbit FITC labeled antibody, which recognize CYP1A1/1A2. The cells were washed again and then incubated with secondary anti-mouse TRITC labeled antibody, which recognized β-III tubulin or GFAP. The cells were then mounted with the mounting media and visualized under fluorescence microscope. Experiments were performed at least three times, and, on average, 20 fields were evaluated for double blind scoring on each slide. RT-PCR analysis Total RNA was extracted from cultured brain cells treated with and without 3-MC by Trizol LS (Life Technologies, U.S.A.) according to the described protocol (Kapoor et al., 2006). cDNA was synthesized as described in our earlier study (Kapoor et al., 2006) and used for subsequent PCR. Prior to the amplification of CYP1A isoenzymes, normalization was carried out with glyceraldehyde 3-phosphate dehydrogenase (GAPDH), the housekeeping gene. PCR reactions for CYP1A1, 1A2 and GAPDH were carried out as described by Johri et al. (2006). PCR products were analysed in VERSA DOC Imaging system, Model 1000 (Biorad, USA). Densitometric analysis of the bands was carried out using Quantity One Quantitation Software version 4.3.1 (Biorad, U.S.A).
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Table 2 Effect of polyclonal antibody raised against rat liver CYP1A1/1A2 on EROD a activity in cultured rat brain neuronal and glial cells Neuronal Cells Control None 11.26 ± 2.84 Preimmune IgG 0.15 mg 11.0 ± 2.5 0.30 mg 10.5 ± 2.0 0.80 mg 9.8 ± 1.0 Anti-CYP1A1/1A2 0.15 mg 8.2 ± 1.0 0.30 mg 6.1 ± 1.2 0.80 mg 4.0 ± 0.7 b
Glial Cells Treated
Control
Treated
18.54 ± 3.15
4.37 ± 1.00
11.67 ± 1.50
17.8 ± 1.8 16.9 ± 1.5 16.3 ± 1.3
9.31 ± 1.1 8.52 ± 1.2 7.52 ± 1.1
11.2 ± 1.9 10.6 ± 1.3 9.9 ± 1.2
8.04 ± 1.3 b 5.10 ± 0.7 b 2.00 ± 0.3 b
6.99 ± 1.0 5.54 ± 0.9 3.76 ± 0.5 b
4.82 ± 0.80 b 2.65 ± 0.04 b 0.92 ± 0.05 b
All values are mean ± S.E. of 3 experiments. a Specific activity is expressed in pmoles resorufin/min/ mg protein. b p b 0.05 when compared to the controls.
immunofluorescence in these glial enriched cultures (data not shown). Cytotoxicity assessment and EROD activity in brain cells Microsomes from untreated cultured neuronal and glial cells were found to catalyze the CYP1A1/1A2 dependent EROD activity. The specific activity in control neuronal cells was
Statistical analysis Student's t-test was employed to calculate the statistical significance between control and treated groups. p b 0.05 was considered to be significant. Results Assessment of purity of neuronal and glial cells by immunocytochemistry Simultaneous staining of the neuronal cells with β-III tubulin (neuronal marker) and GFAP (glial), gave positive immunofluorescence for β-III tubulin (90–95%), whereas GFAP staining was only 5% in these neuronal enriched cultures. Similarly, when the glial cells were simultaneously stained with β-III tubulin and GFAP, positive immunofluorescence was seen for GFAP (90–95%), whereas, β-III tubulin showed only 5%
Table 1 EROD a activity in cultured neuronal and glial cells treated with 1.0 μM 3methylchlorantherene for 48 h
Control Treated
Neuronal cells
Glial cells
11.26 ± 2.84 18.54 ± 3.15 b
4.37 ± 1.00 11.67 ± 1.50 b
All values are mean ± S.E. of 3 experiments. a Specific activity in pmoles resorufin/min/mg protein. b p b 0.05 when compared to the controls.
Fig. 1. a) Ethidium bromide stained agarose gel showing rat GAPDH mRNA in cultured neuronal and glial cells. Lane 1 contains 5 μl of the RT-PCR product without RNA. Lanes 2 and 3 contain 5 μl of the RT-PCR product isolated from control and 3-MC treated glial cells respectively. Lanes 4 and 5 contain 5 μl of the RT-PCR product of RNA isolated from control and 3-MC treated neuronal cells respectively. Lanes 6 and 7 contain 5 μl of the RT-PCR product isolated from control liver and 1.0 Kb DNA ladder respectively. b) Densitometric analysis of RT-PCR products (mean ± 3 gels). G and N correspond to the intensity of RT-PCR product of RNA isolated from cultured glial and neuronal cells respectively.
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approximately 2 fold greater as compared to the specific activity in control glial cells (Table 1). When the neuronal and glial cells were exposed to different concentrations of 3-MC for different time periods and assessed by the MTT assay for cytotoxicity, it was found that none of the concentrations used were toxic at any time point (data not shown). An increase in the activity of EROD was also observed in the cultured brain cells with increase in the concentration of 3-MC and with the increase in the duration of exposure (data not shown). However, based on the linear response and to compare the sensitivity of the cultured neuronal or glial cells to 3-MC under identical conditions, a concentration of 1.0 μM and time interval of 48 h were chosen for the further induction studies. Addition of 3-MC (1.0 μM) for 48 h was found to produce a significant increase in the EROD activity in cultured neuronal and glial cells. The percentage of induction in the glial cells was more than that observed in neuronal cells (Table 1). Effect of anti-CYP1A1/1A2 on cultured rat brain cell EROD activity Addition of polyclonal antibody raised against rat liver CYP1A1/1A2 isoenzymes to the microsomes isolated from control neuronal and glial cells did not produce any significant inhibition in the EROD activity at lower concentrations. Sig-
Fig. 3. a) Ethidium bromide stained agarose gel showing rat CYP1A2 mRNA in cultured neuronal and glial cells. Lane 1 contains 5 μl of the RT-PCR product without RNA. Lanes 2 and 3 contains 5 μl of the RT-PCR product isolated from control and 3-MC treated glial cells. Lanes 4 and 5 contain 5 μl of the RT-PCR product of RNA isolated from control and 3-MC treated neuronal cells. Lanes 6 and 7 contain 5 μl of the RT-PCR product isolated from control liver and 1.0 Kb DNA ladder respectively. b) Densitometric analysis of RT-PCR products (mean ± 3 gels). G and N correspond to the intensity of RT-PCR product of RNA isolated from cultured glial and neuronal cells respectively.
nificant inhibition in the microsomal EROD activity was observed only at the highest concentration of the antibody used (Table 2). Both, cultured neuronal or glial cells exhibited almost similar magnitude of inhibition in the enzyme activity on addition of the polyclonal anti-CYP1A1/1A2 to the reaction mixture (Table 2). Addition of preimmune IgG had no significant effect when added in vitro to the microsomes isolated from control neuronal or glial cells (Table 2). In vitro addition of antiCYP1A1/1A2 to the microsomes isolated from 3-MC pretreated neuronal or glial cells produced a significant concentration dependent inhibition of the enzyme activity at all the concentrations studied with approximately 90% inhibition of the enzyme activity occurring at the highest concentration of the antibody (Table 2). Reverse transcriptase-PCR analysis
Fig. 2. a) Ethidium bromide stained agarose gel showing rat CYP1A1 mRNA in cultured neuronal and glial cells. Lane 1 contains 5 μl of the RT-PCR product without RNA. Lanes 2 and 3 contain 5 μl of the RT-PCR product isolated from control and 3-MC treated glial cells respectively. Lanes 4 and 5 contain 5 μl of the RT-PCR product of RNA isolated from control and 3-MC treated neuronal cells respectively. Lanes 6 and 7 contain 5 μl of the RT-PCR product isolated from control liver and 1.0 Kb DNA ladder respectively. b) Densitometric analysis of RT-PCR products (mean ± 3 gels). G and N correspond to the intensity of RT-PCR product of RNA isolated from cultured glial and neuronal cells respectively.
Reverse transcriptase-PCR analysis with primers specific for rat liver GAPDH resulted in the formation of PCR products of expected band size of 194 bp in the RNA isolated from the rat brain neuronal and glial cells cultured in DMEM alone or in DMEM plus 3-MC (Fig. 1). Densitometric analysis of the PCR products revealed that PCR amplification resulted in the formation of products of almost equal intensity (Fig. 1). Using primers specific for CYP1A1 and CYP1A2, PCR analysis of the RT product obtained from the RNA isolated from rat brain neuronal and glial cells cultured in DMEM alone or in DMEM
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Fig. 4. Immunocytochemical detection of CYP1A1 in cultured rat brain neuronal cells. a, b and c represent primary cultures of neuronal cells in DMEM. d, e and f represent cultures of neuronal cells in DMEM + MC. a and d show cells in culture that are positive for β-III tubulin (red-TRITC), a neuronal marker. b and e show immunoreactivity in the same neuronal cells with anti-CYP1A1 (green-FITC). c and f represent an overlay of the two images control and MC treated respectively. Original magnification ×40, scale 20 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 5. Immunocytochemical detection of CYP1A1 in cultured rat brain glial cells. a, b and c represent primary cultures of glial cells in DMEM. d, e and f represent cultures of glial cells in DMEM + MC. a and d show cells in culture that are positive for anti-GFAP (red-TRITC), a glial marker. b and e show immunoreactivity in the same glial cells with anti-CYP1A1 (green-FITC). c and f represent an overlay of the two images control and MC treated respectively. Original magnification ×40, scale 20 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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plus 3-MC, after normalization with GAPDH produced a distinct band visible by agarose gel electrophoresis and of the correct size of 341 bp and 793 bp respectively (Figs. 2 and 3). PCR products of similar size were obtained from the RNA isolated from control rat liver, using the same primers. The basal expression of CYP1A1 and CYP1A2 mRNA was more in cultured neuronal cells as compared to the glial cells. Treatment of the cultured brain cells with MC produced a significant increase in CYP1A1 and CYP1A2 mRNA expression in both neuronal and glial cells, relative to the corresponding untreated cells. However, densitometric analysis revealed that the percentage of induction was more in glial cells as compared to the neuronal cells. CYP1A1 was found to be induced to a greater extent as compared to CYP1A2 (Figs. 2 and 3). Western blot analysis Immunoblot analysis of the solubilized microsomal proteins isolated from the untreated or treated neuronal and glial cells with hepatic anti-CYP1A1/2A2 did not exhibit detectable immunoreactivity co-migrating with the liver isoenzyme in either control rat brain cells or cells cultured in the presence of 3-MC (data not shown). Assessment of CYP1A1/1A2 expression in cultured neuronal and glial cells by immunocytochemistry Both neuronal and glial cells cultured with DMEM alone or treated with MC, grown on PLL coated glass slides, when fixed, and stained with anti-CYP1A1 antibody and with anti-β-III tubulin or anti-GFAP, and the secondary antibodies labeled with FITC or TRITC showed positive staining for CYP1A1. As shown in Fig. 4, all the neuronal cells exhibiting staining with anti-β-III tubulin also expressed CYP1A1 as judged by similar pattern of staining with anti-CYP1A1. Likewise, all glial cells exhibiting staining with anti-GFAP also expressed CYP1A as judged by staining with anti-CYP1A (Fig. 5). 3-MC treatment was not found to have any effect on the immunoreactivity observed with anti-GFAP or anti-β-III tubulin alone in either glial or neuronal cells (Figs. 4 and 5). As monoclonal antiGFAP used recognizes astrocytes and Bergman glial cells only, some of the cells immunopositive for CYP1A are negative for anti-GFAP, as it is a mixed glial culture from whole brain, which may also contain the oligodendrocytes. Similarly, some anti-βIII tubulin negative cells, which were immunopositive for CYP1A, could be the endothelial or fibroblast cells contaminating the neuronal cultures. Software analysis (Leica Qfluro Standard, Leica Microsystems Imaging Solutions Ltd., Version V1.2.0) revealed that treatment of 3-MC resulted in an increase in the intensity of FITC fluorescence. The mean intensity of FITC in treated neuronal cells was increased (27%) as compared to the control cells (Fig. 4). Likewise the glial cells exhibited an increased (174%) intensity of fluorescence of treated cells when compared to the control cells (Fig. 5). Moreover, the percentage of induction was more in glial cells than that observed in neuronal cells.
Discussion Expression of CYP1A isoenzymes and their catalytic activity in cultured brain cells is consistent with the recent studies indicating the presence of arylhydrocarbon receptor (Ahr) system in primary cultures of rat neuronal and glial cells (Filbrandt et al., 2004; Pravettoni et al., 2005). Kainu et al. (1995) have also earlier reported the localization of the main components of the Ahr pathway in the neurons of several brain areas of adult male rats. Several fold increase in the EROD activity as well as the expression of CYP1A isoenzymes in the cultured brain cells following treatment of 3-MC have indicated that responsiveness of CYP1A isoenzymes to CYP inducers is retained in the primary cultures of brain cells. Though earlier studies have demonstrated that glial cells harvested from rat brain tissues respond to CYP1A1/1A2 inducers, not much information is available about the responsiveness of cultured neurons (Legare et al., 1997; Filbrandt et al., 2004). Interestingly, cultured neurons exhibit relatively higher activity of EROD than the glial cells, though the magnitude of induction in the enzyme activity after in vitro addition of 3-MC was less in the neurons when compared to the glial cells. Immunocytochemistry, using specific antibodies for CYP1A1/ 1A2 and RT-PCR, with primers specific for rat liver CYP1A1and CYP1A2, have also shown higher immunoreactivity and mRNA expression of CYP1A1 and CYP1A2 in cultured neurons than the glial cells. However, the inability to detect CYP1A1/1A2 protein expression by immunoblotting in either control or 3-MC treated cultured neuronal and glial cells could be because of the relatively lower protein expression of CYP1A1/1A2 in brain cells which maybe beyond the levels of detection of the antibody used in the present study. Filbrandt et al. (2004) have also reported that because of relatively lesser expression of CYP1A1 in astrocytes, CYP1A1 expression and inducibility is not detected at the protein level by immunoblotting. However, induction of CYP1A1 protein expression has been shown in rat C6 glial cells with TCDD treatment (Takanaga et al., 2001). This discrepancy could be attributed to the use of a glial cell line rather than the primary cultures of brain cells used in the present study or reported earlier (Filbrandt et al., 2004). In contrast, the ability to detect responsiveness of cultured brain cells to 3-MC by immunocytochemistry or PCR could be because of the enhanced sensitivity of these assays where even the increase in the expression of single cell could be detected as compared to identifying increase in protein expression from a pool of microsomal proteins by western blotting. Relatively higher activity of CYP1A1/1A2 in cultured neurons have further provided support to the hypothesis that xenobiotic metabolizing CYPs have a physiological role in brain (Hedlund et al., 2001; Nissbrandt et al., 2001; Strobel et al., 2001; Miksys and Tyndale, 2002; Parmar et al., 2003; Gervasini et al., 2004). Konstandi et al. (2005), using selected agonists and antagonists have identified the role of α and β-adrenoreceptors in the regulation of CYP1A1 induction in brain. Adrenoreceptors were found to be involved in the regulation of the CYP1A1 gene at mRNA level. Both, reduced noradrenaline release in CNS and central catecholamine depletion
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resulted in downregulation of CYP1A1 expression (Konstandi et al., 2004, 2005). Recent studies have indicated a role of CYPs in neurotransmission and possible endogenous substrates for CYP2E1, CYP1A2 and CYP2D6 have been identified in the brain. A role of CYP1A isoenzymes in the hydroxylation of estradiol, which is involved in dopamine receptor supersenstivity, has also been demonstrated (Tsuchiya et al., 2005). Although the exact role of CYPs in human brain needs to be elucidated, modulation of CYP1A2 microsomal enzyme activity by serotonin and tryptamine, the two indolamines have lead to the speculation that neurotransmitters and other substances whose local concentrations are finely tuned by several mechanisms, might play a regulatory role on brain CYP1A2 activity (Agundez et al., 1998; Martinez et al., 2000). This local regulatory role of endogenous substances might also possibly help in explaining the relatively higher degree of induction in enzyme activity (EROD) in brain cells, as observed in the present study after the addition of MC, rather than the protein expression. Poor correlation in liver samples between CYP1A2 content, as measured by western immunoblotting and CYP1A2 activity as measured with specific substrates has been reported in the liver samples of patients undergoing surgery (Lucas et al., 1993). This lack of correlation was not attributed to smoking habits or alcohol intake but rather to the influence of local factors or possible endogenous substrates that modulate enzyme activity rather than enzyme expression. Interestingly, relatively higher magnitude of induction of CYP1A isoenzymes in cultured glial cells could be of significance as these cells are the main cellular components of the blood–brain barrier (BBB). As glial cells have an important physiological role in integrating neuronal inputs, neurotransmitter release and the protection and repair of nervous tissue, the greater responsiveness of CYP1A isoenzymes to CYP1Ainducers in glial cells could be attributed to their role in toxication–detoxication mechanisms. The greater magnitude of induction of CYP1A isoenzymes in glial cells has further provided support to the assumption that glial cells function as a control element for the xenobiotics, which reach the blood– brain barrier. Earlier studies have shown that stimulation of astrocytes, the major glial cells in the CNS, with mediators of inflammation results in downregulation of CYP1A activity in cultured astrocytes (Nicholson and Renton, 1999, 2002; Abdulla and Renton, 2005). Recent evidences have shown that several CYP isoforms including CYP1A found in the brain are depressed during a localized CNS inflammatory response (Nicholson and Renton, 2001, 2002). However, β-adrenoreceptor stimulation during conditions of CNS inflammation prevented the downregulation in CYP1A1 and CYP1A2 activity in astrocytes, demonstrating the protective role of β-adrenoreceptors during CNS inflammation (Abdulla and Renton, 2005). In conclusion, the present study clearly demonstrates the expression of CYP1A1 and CYP1A2 in both the cultured neuronal and glial cells. Higher levels of these isoforms in cultured brain cells support the physiological role of CYP1A in brain. The greater magnitude of induction of CYP1A in glial cells is of significance, as these cells are components of the
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blood–brain barrier and it is suggested that they have a potential role in the toxication–detoxication mechanism. However, induction of CYP1A in neurons could be of toxicological significance, as alterations in the levels of this CYP isoforms could also result in modulating the physiological effects regulated by these isoenzymes in brain. Acknowledgement Authors are grateful to Director, ITRC for his keen interest in the work. The financial assistance of the Council of Science & Technology, U.P and CSIR, India for carrying out the above studies is gratefully acknowledged. The technical assistance of Mr. B. S. Pandey and Mr. Rajesh Misra and computer help of Mr. Mohd. Aslam is gratefully acknowledged. ITRC Publication No.: 2482. References Abdulla, D., Renton, K.W., 2005. Beta-adrenergic receptor modulation of the LPS-mediated depression in CYP1A activity in astrocytes. Biochemical Pharmacology 69, 741–750. Agundez, J.A., Gallardo, L., Martinez, C., Gervasini, G., Benitez, J., 1998. Modulation of CYP1A2 enzyme activity by indoleamines: inhibition by serotonin and tryptamine. Pharmacogenetics 8, 251–258. Anandatheerthavarada, H.K., Shankar, S.K., Ravindranath, V., 1990. Rat brain cytochromes P-450: catalytic, immunochemical properties and inducibility of multiple forms: Preparation of brain microsomes with cytochrome P450 activity using calcium aggregation method NADPH cytochrome P-450 reductase in rat, mouse and human brain. Brain Research 536, 339–343. Cherng, S.H., Lin, P., Yang, J.L., Hsu, S.L., Lee, H., 2001. Benzo[g,h,i]perylene synergistically transactivates benzo[a]pyrene-induced CYP1A1 gene expression by aryl hydrocarbon receptor pathway. Toxicology and Applied Pharmacology 170, 63–68. Eaton, D.L., Gallagher, E.P., Bammler, T.K., Kunze, K.L., 1995. Role of cytochrome P450 1A2 in chemical carcinogenesis: implications for human variability in expression and enzyme activity. Pharmacogenetics 5, 259–274. Filbrandt, C.R., Wu, Z., Zlokovic, B., Opanashuk, L., Gasiewicz, T.A., 2004. Presence and functional activity of the aryl hydrocarbon receptor in isolated murine cerebral vascular endothelial cells and astrocytes. Neurotoxicology 25, 605–616. Gervasini, G., Carrillo, J.A., Benitez, J., 2004. Potential role of cerebral cytochrome P450 in clinical pharmacokinetics: modulation by foreign chemicals. Clinical Pharmacokinetics 43, 693–706. Gonzalez, F.J., Matsunaga, T., Nagata, K., 1989. Structure and regulation of P450s in the rat P450IIA gene subfamily. Drug Metabolism Reviews 20, 827–837. Guengerich, F.P., 1990. Characterization of roles of human cytochrome P450 enzymes in carcinogen metabolism. Asia Pacific Journal of Pharmacology 5, 327–345. Hedlund, E., Gustafsson, J.A., Warner, M., 2001. Cytochrome P450 in the brain; a review. Current Drug Metabolism 2, 245–263. Hodgson, A.V., White, T.B., White, J.W., Strobel, H.W., 1993. Expression analysis of the mixed function oxidase system in rat brain by the polymerase chain reaction. Molecular and Cellular Biochemistry 120, 171–179. Johri, A., Dhawan, A., Singh, R.L., Parmar, D., 2006. Effect of prenatal exposure of deltamethrin on the ontogeny of xenobiotics metabolizing cytochrome P450s in the brain and liver of offsprings. Toxicology and Applied Pharmacology 214, 279–289. Kainu, T., Gustafsson, J.A., Pelto-Huikko, M., 1995. The dioxin receptor and its nuclear translocator (Arnt) in the rat brain. Neuroreport 6, 2557–2560. Kapitulnik, J., Gelboin, H.V., Guengerich, F.P., Jacobowitz, D.M., 1987. Immunohistochemical localization of cytochrome P-450 in rat brain. Neuroscience 20, 829–833.
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Kapoor, N., Pant, A.B., Dhawan, A., Dwievedi, U.N., Gupta, Y.K., Seth, P.K., Parmar, D., 2006. Differences in sensitivity of cultured rat brain neuronal and glial cytochrome P450 2E1 to ethanol. Life Sciences (May 7, Electronic publication ahead of print). Kohler, C., Eriksson, L.G., Hansson, T., Warner, M., Ake-Gustafsson, J., 1988. Immunohistochemical localization of cytochrome P-450 in the rat brain. Neuroscience Letters 84, 109–114. Konstandi, M., Johnson, E.O., Marselos, M., Kostakis, D., Fotopoulos, A., Lang, M.A., 2004. Stress-mediated modulation of B(alpha) P-induced hepatic CYP1A1: role of catecholamines. Chemical Biological Interactions 147, 65–77. Konstandi, M., Kostakis, D., Harkitis, P., Marselos, M., Johnson, E.O., Adamidis, K., Lang, M.A., 2005. Role of adrenoceptor-linked signaling pathways in the regulation of CYP1A1 gene expression. Biochemical Pharmacology 69, 277–287. Legare, M.E., Hanneman, W.H., Barhoumi, R., Tiffany-Castiglioni, E., 1997. The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure in primary rat astroglia: identification of biochemical and cellular targets. Neurotoxicology 18, 515–524. Lucas, D., Berthou, F., Girre, C., Poitrenaud, F., Menez, J.F., 1993. Highperformance liquid chromatographic determination of chlorzoxazone and 6hydroxychlorzoxazone in serum: a tool for indirect evaluation of cytochrome P450 2E1 activity in humans. Journal of Chromatography 622, 79–86. Martinez, C., Gervasini, G., Agundez, J.A., Carrillo, J.A., Ramos, S.I., GarciaGamito, F.J., Gallardo, L., Benitez, J., 2000. Modulation of midazolam 1hydroxylation activity in vitro by neurotransmitters and precursors. European Journal of Clinical Pharmacology 56, 145–151. Miksys, S.L., Tyndale, R.F., 2002. Drug metabolizing cytochrome P450s in the brain. Journal of Psychiatry Neuroscience 27, 406–415. Miksys, S.L, Hoffman, E., Tyndale, R.F., 2000. Regional and cellular induction of nicotine metabolizing CYP2B1 in rat brain by chronic nicotine treatment. Biochemical Pharmacology 59, 1501–1511. Nebert, D.W., Jones, J.E., 1989. Regulation of the mammalian cytochrome P1450 (CYP1A1) gene. International Journal of Biochemistry 21, 243–252. Nelson, D.R., Koymans, L., Kamataki, T., Stegeman, J.J., Feyereisen, R., Waxman, D.J., Waterman, M.R., Gotoh, O., Coon, M.J., Estrabrook, R.W., Gunsalus, I.C., Nebert, D.W., 1996. P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature. Pharmacogenetics 6, 1–42. Nicholson, T.E., Renton, K.W., 1999. Modulation of cytochrome P450 by inflammation in astrocytes. Brain Research 827, 12–18. Nicholson, T.E., Renton, K.W., 2001. Role of cytokines in the lipopolysaccharide-evoked depression of cytochrome P450 in the brain and liver. Biochemical Pharmacology 62, 1709–1717.
Nicholson, T.E., Renton, K.W., 2002. The role of cytokines in the depression of CYP1A activity using cultured astrocytes as an in vitro model of inflammation in the central nervous system. Drug Metabolism Disposition 30, 42–46. Nissbrandt, H., Bergquist, F., Jonason, J., Engberg, G., 2001. Inhibition of cytochrome P450 2E1 induces an increase in extracellular dopamine in rat substantia nigra: a new metabolic pathway? Synapse 40, 294–301. Omiecinski, C.J., Redlich, C.A., Costa, P., 1990. Induction and developmental expression of cytochrome P450 IA1 messenger RNA in rat and human tissues: detection by the polymerase chain reaction. Cancer Research 50, 4315–4321. Parmar, D., Dhawan, A., Seth, P.K., 1998. Immunochemical evidence for the presence of phenobarbital (PB) and 3-methylcholanthrene (MC) inducible cytochrome P450 isoenzymes in rat brain. International Journal of Toxicology 17, 619–630. Parmar, D., Dayal, M., Seth, P.K., 2003. Expression of cytochrome P450s (P450s) in brain: physiological, pharmacological and toxicological consequences. Proceedings of Indian National Academy of Sciences, vol. 6, pp. 905–928. Pravettoni, A., Colciago, A., Negri-Cesi, P., Villa, S., Celotti, F., 2005. Ontogenetic development, sexual differentiation, and effects of Aroclor 1254 exposure on expression of the arylhydrocarbon receptor and of the arylhydrocarbon receptor nuclear translocator in the rat hypothalamus. Reproductive Toxicology 20, 521–530. Schilter, B., Omiecinski, C.J., 1993. Regional distribution and expression modulation of cytochrome P-450 and epoxide hydrolase mRNAs in the rat brain. Molecular Pharmacology 44, 990–996. Strobel, H.W., Thompson, C.M., Antonovic, L., 2001. Cytochromes P450 in brain: function and significance. Current Drug Metabolism 2, 199–214. Takanaga, H., Kunimoto, M., Adachi, T., Tohyama, C., Aoki, Y., 2001. Inhibitory effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on cAMP-induced differentiation of rat C6 glial cell line. Journal of Neuroscience Research 64, 402–409. Tsuchiya, Y., Nakajima, M., Yokoi, T., 2005. Cytochrome P450-mediated metabolism of estrogens and its regulation in human. Cancer Letters 227, 115–124. Warner, M., Kohler, C., Hansen, T., Gustafsson, J.A., 1988. Regional distribution of cytochrome P450 in the rat brain: spectral quantification and contribution of P450b,e and P450c,d. Journal of Neurochemistry 50, 1057–1065.
Cytochrome P450 1A isoenzymes in brain cells: Expression and inducibility in cultured rat brain neuronal and glial cells Nidhi Kapoor a , Aditya B. Pant b , Alok Dhawan b , Uppendra N. Dwievedi a , Prahlad K. Seth b,1 , Devendra Parmar b,⁎ b
a Biochemistry Department, Lucknow University, University Road, Lucknow, India Developmental Toxicology Division, Industrial Toxicology Research Centre, P.O. Box 80, Mahatma Gandhi Marg, Lucknow-226 001, India
Received 15 June 2006; accepted 1 August 2006
Abstract Studies initiated to determine the expression of CYP1A1/1A2 isoenzymes in the primary cultures of rat brain neuronal and glial cells revealed significant activity of CYP1A-dependent 7-ethoxyresorufin-o-dealkylase (EROD) in microsomes prepared from both rat brain neuronal and glial cells. RT-PCR and immunocytochemical studies demonstrated constitutive mRNA and protein expression of CYP1A1 and 1A2 isoenzymes in cultured neuronal and glial cells. Cultured neurons exhibited relatively higher constitutive mRNA and protein expression of CYP1A1 and 1A2 isoenzymes, associated with higher activity of EROD than the glial cells. Induction studies with 3-methylchlorantherene (MC), a known CYP1Ainducer, resulted in significant concentration dependent increase in the activity of EROD in cultured rat brain cells with glial cells exhibiting a greater magnitude of induction than the neuronal cells. This difference in the increase in enzyme activity was also observed with RT-PCR and immunocytochemical studies, indicating relatively higher increase in CYP1A1 and 1A2 mRNA as well as protein expression in the cultured glial cells when compared to the neuronal cells. The greater magnitude of induction of CYP1A1 in glial cells is of significance, as these cells are components of the blood–brain barrier and it is suggested that they have a potential role in the toxication–detoxication mechanism. Our data indicating differences in the expression and sensitivity of CYP1A1 isoenzymes in cultured rat brain cells will not only help in identifying and distinguishing xenobiotic metabolizing capability of these cells but also in understanding the vulnerability of these specific cell types towards neurotoxicants. © 2006 Elsevier Inc. All rights reserved. Keywords: Cytochrome P450 1A; Neurons; Glia; Induction; mRNA; Enzyme
Introduction Cytochrome P450 (CYP) is a heme containing superfamily of enzymes that metabolize a broad spectrum of endogenous and exogenous compounds (Nelson et al., 1996). CYP1A (CYP1A1 and 1A2) isoenzymes exhibit tissue specificity in constitutive as well as inducible expression. CYP1A1, considered to be predominantly constitutively expressed in extrahepatic tissues, is inducible in liver and extrahepatic tissues while ⁎ Corresponding author. Tel.: +91 522 2613786/2627586x261/262; fax: +91 522 2628227, 2611547. E-mail address: [email protected] (D. Parmar). 1 Present Address: Biotech Park, Sector-G, Jankipuram, Kursi Road, Lucknow, India. 0024-3205/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2006.08.002
CYP1A2, generally known to be a predominantly hepatic form, exhibits both constitutive and inducible expression (Eaton et al., 1995; Guengerich, 1990). CYP1A1 and 1A2 are of toxicological importance because of their role in the bioactivation of environmental toxins and carcinogens such as aromatic amines and polycyclic aromatic hydrocarbons (PAH; Gonzalez et al., 1989; Nebert and Jones, 1989; Omiecinski et al., 1990; Cherng et al., 2001). RT-PCR and immunoblotting studies have revealed the constitutive presence of CYP1A1 and CYP1A2 in rat brain (Kohler et al., 1988; Warner et al., 1988; Anandatheerthavarada et al., 1990; Hodgson et al., 1993; Schilter and Omiecinski, 1993). The marked structural complexity and cellular heterogeneity of brain was reflected in the regional and cellular differences observed in the distribution of CYP1A1/1A2 (Kapitulnik et al.,
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1987; Kohler et al., 1988). Spatial distribution of CYP1A1/1A2 in the different cell types of the brain has indicated endogenous functions. Although the endogenous substrates of CYP1A1/ 1A2 are yet to be identified, the activity of CYP1A isoenzymes is known to be modulated by several neurotransmitters (Agundez et al., 1998). Furthermore, endogenous processes such as a localized inflammatory response in the brain can also modulate its levels, thereby influencing drug response. For tissues exhibiting low regenerative capacity, such as the brain, such modulation would be of major toxicological significance. Although CYPs in brain occur at only 1–5% of the levels of the liver enzyme, it has been reported that the levels of CYPs in specific neurons can be as high or even higher than the levels in hepatocytes (Miksys et al., 2000; Strobel et al., 2001; Parmar et al., 2003; Gervasini et al., 2004). In an attempt to further investigate the distribution of CYPs within the brain, the present study aimed to determine the expression and catalytic activity of CYP1A1 and 1A2 isoenzymes in primary cultured rat brain neuronal (N) and glial cells (G), the two major cell types of the brain. Furthermore, the responsiveness of these cultured rat brain neuronal and glial cells to 3-methylchlorantherene (MC), a CYP1A1/1A2 inducer, was also studied to ascertain the suitability of using these cultured brain cells as a tool for understanding the mechanisms of neurotoxicity and neurodegeneration.
neurons were attached to the PLL coated surface within about 5 min. Unattached cells were then transferred to second and third set of PLL coated flask after which the unattached glial cells were transferred to a fresh PLL coated flask. Only the first and the last batches of flasks were used as a source of N and G cells respectively. Assessment of purity of neuronal and glial cells by immunocytochemistry The purity of neuronal and glial cultures was assessed as described by Kapoor et al. (2006). In brief, the cultured cells were fixed, permeabilized and then incubated with primary antibody (anti-β-III tubulin or anti-GFAP). The cells were then washed and incubated with anti-rabbit FITC labeled secondary antibody, which recognize GFAP, for 1 h. The cells were then washed again and incubated with secondary anti-mouse TRITC labeled antibody, which recognize β-III tubulin. The cells were then mounted with the mounting media and visualized and analyzed under fluorescence microscope (Leica, Germany) using Leica Q Fluoro Software, respectively. The purity of N and G cells preparation, identified by counting multiple fields after staining with cell specific markers, was 90–95%. Treatment of brain cells and cytotoxicity assessment
Materials and methods Chemicals Monoclonal antibodies for GFAP (Chemicon International) and β-III tubulin (Concave company, California) and Secondary Fluorescence antibodies FITC and Rhodamine (TRITC), (Jackson Imunoresearch laboratories, Inc.) were kindly gifted by Dr. Eugene Major, National Institute of Neurological Disorders and Stroke (NINDS), Bethesda, Maryland, U.S.A. Antirat hepatic CYPlAl/1A2 was obtained from Chemicon, USA. All the other chemicals including PCR enzymes and culture reagents were procured from M/s Sigma-Aldrich, St. Louis, MI, U.S.A or Life Technologies, USA. Neuronal and glial cell culture Pregnant albino Wistar rats weighing 175–200 gm (∼ 8 week old) were obtained from the Industrial Toxicology Research Centre breeding colony and raised on a commercial pellet diet and water ad libitum. Animals were cared for in accordance to the policy laid down by the Animal Care Committee of Industrial Toxicology Research Centre and the Ethical Committee of the Centre approved Animal Experimentation. For neuronal cell culture, 14 days old embryos and for glial cells, 0–1 day old pups were used as described in our earlier study by Kapoor et al. (2006). In brief, brain tissue was minced, trypsinised and incubated with DMEM media containing 12% FBS. Cells were then dissociated and the resulting cell suspension were filtered through a 50 μm diameter nylon mesh and pelleted by centrifugation. Approximately, 30 million cells in 12–15 ml medium were plated in each PLL coated flask. More than 90% of the
The cultured cells were treated with different concentrations (0.5-, 1.0-, 2.0-, 3.0-, 4.0- and 8.0 μM) of 3-methychlorantherene (3-MC) for different time intervals (12-, 24-, 48-, 72- and 96 h). Cytotoxicity was assessed by a colorimetric assay based on the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) dye as described by Kapoor et al. (2006). In brief, after the exposure of 3-MC, MTT was added to each well and left for 4-6 h. The medium containing the MTT was aspirated carefully and 150 μl of DMSO was added to each well and incubated for 30 min. The solution in each well was mixed well and the absorbance read on a microplate reader at 550 nm. Enzyme activity The neuronal and glial cells, after treatment with 3-MC were processed for isolation of microsomes as described in our earlier study (Kapoor et al., 2006). The activity of 7-ethoxyresorufinO-deethylase (EROD) was determined in cultured rat brain neuronal and glial cell microsomes by the method of Parmar et al. (1998) using a Perkin Elmer LS 55 Luminescence spectrometer. As isoforms other than CYP1A1 may constitute CYPs in control brain (Warner et al., 1988; Parmar et al., 2003), antibody inhibition studies with anti-CYP1A1/1A2 were also carried out to further identify the role of CYP1A1/1A2 in catalyzing the activity of EROD in brain cells as described earlier (Parmar et al., 1998). Immunocytochemistry For immunocytochemistry, the cultured neuronal and glial cells were fixed, permeabilized and incubated with primary
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antibody for anti-β-III tubulin, anti-CYP1A1/1A2, or antiGFAP and anti-CYP1A1/1A2 as described by Kapoor et al. (2006). The cells were then washed and incubated with antirabbit FITC labeled antibody, which recognize CYP1A1/1A2. The cells were washed again and then incubated with secondary anti-mouse TRITC labeled antibody, which recognized β-III tubulin or GFAP. The cells were then mounted with the mounting media and visualized under fluorescence microscope. Experiments were performed at least three times, and, on average, 20 fields were evaluated for double blind scoring on each slide. RT-PCR analysis Total RNA was extracted from cultured brain cells treated with and without 3-MC by Trizol LS (Life Technologies, U.S.A.) according to the described protocol (Kapoor et al., 2006). cDNA was synthesized as described in our earlier study (Kapoor et al., 2006) and used for subsequent PCR. Prior to the amplification of CYP1A isoenzymes, normalization was carried out with glyceraldehyde 3-phosphate dehydrogenase (GAPDH), the housekeeping gene. PCR reactions for CYP1A1, 1A2 and GAPDH were carried out as described by Johri et al. (2006). PCR products were analysed in VERSA DOC Imaging system, Model 1000 (Biorad, USA). Densitometric analysis of the bands was carried out using Quantity One Quantitation Software version 4.3.1 (Biorad, U.S.A).
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Table 2 Effect of polyclonal antibody raised against rat liver CYP1A1/1A2 on EROD a activity in cultured rat brain neuronal and glial cells Neuronal Cells Control None 11.26 ± 2.84 Preimmune IgG 0.15 mg 11.0 ± 2.5 0.30 mg 10.5 ± 2.0 0.80 mg 9.8 ± 1.0 Anti-CYP1A1/1A2 0.15 mg 8.2 ± 1.0 0.30 mg 6.1 ± 1.2 0.80 mg 4.0 ± 0.7 b
Glial Cells Treated
Control
Treated
18.54 ± 3.15
4.37 ± 1.00
11.67 ± 1.50
17.8 ± 1.8 16.9 ± 1.5 16.3 ± 1.3
9.31 ± 1.1 8.52 ± 1.2 7.52 ± 1.1
11.2 ± 1.9 10.6 ± 1.3 9.9 ± 1.2
8.04 ± 1.3 b 5.10 ± 0.7 b 2.00 ± 0.3 b
6.99 ± 1.0 5.54 ± 0.9 3.76 ± 0.5 b
4.82 ± 0.80 b 2.65 ± 0.04 b 0.92 ± 0.05 b
All values are mean ± S.E. of 3 experiments. a Specific activity is expressed in pmoles resorufin/min/ mg protein. b p b 0.05 when compared to the controls.
immunofluorescence in these glial enriched cultures (data not shown). Cytotoxicity assessment and EROD activity in brain cells Microsomes from untreated cultured neuronal and glial cells were found to catalyze the CYP1A1/1A2 dependent EROD activity. The specific activity in control neuronal cells was
Statistical analysis Student's t-test was employed to calculate the statistical significance between control and treated groups. p b 0.05 was considered to be significant. Results Assessment of purity of neuronal and glial cells by immunocytochemistry Simultaneous staining of the neuronal cells with β-III tubulin (neuronal marker) and GFAP (glial), gave positive immunofluorescence for β-III tubulin (90–95%), whereas GFAP staining was only 5% in these neuronal enriched cultures. Similarly, when the glial cells were simultaneously stained with β-III tubulin and GFAP, positive immunofluorescence was seen for GFAP (90–95%), whereas, β-III tubulin showed only 5%
Table 1 EROD a activity in cultured neuronal and glial cells treated with 1.0 μM 3methylchlorantherene for 48 h
Control Treated
Neuronal cells
Glial cells
11.26 ± 2.84 18.54 ± 3.15 b
4.37 ± 1.00 11.67 ± 1.50 b
All values are mean ± S.E. of 3 experiments. a Specific activity in pmoles resorufin/min/mg protein. b p b 0.05 when compared to the controls.
Fig. 1. a) Ethidium bromide stained agarose gel showing rat GAPDH mRNA in cultured neuronal and glial cells. Lane 1 contains 5 μl of the RT-PCR product without RNA. Lanes 2 and 3 contain 5 μl of the RT-PCR product isolated from control and 3-MC treated glial cells respectively. Lanes 4 and 5 contain 5 μl of the RT-PCR product of RNA isolated from control and 3-MC treated neuronal cells respectively. Lanes 6 and 7 contain 5 μl of the RT-PCR product isolated from control liver and 1.0 Kb DNA ladder respectively. b) Densitometric analysis of RT-PCR products (mean ± 3 gels). G and N correspond to the intensity of RT-PCR product of RNA isolated from cultured glial and neuronal cells respectively.
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approximately 2 fold greater as compared to the specific activity in control glial cells (Table 1). When the neuronal and glial cells were exposed to different concentrations of 3-MC for different time periods and assessed by the MTT assay for cytotoxicity, it was found that none of the concentrations used were toxic at any time point (data not shown). An increase in the activity of EROD was also observed in the cultured brain cells with increase in the concentration of 3-MC and with the increase in the duration of exposure (data not shown). However, based on the linear response and to compare the sensitivity of the cultured neuronal or glial cells to 3-MC under identical conditions, a concentration of 1.0 μM and time interval of 48 h were chosen for the further induction studies. Addition of 3-MC (1.0 μM) for 48 h was found to produce a significant increase in the EROD activity in cultured neuronal and glial cells. The percentage of induction in the glial cells was more than that observed in neuronal cells (Table 1). Effect of anti-CYP1A1/1A2 on cultured rat brain cell EROD activity Addition of polyclonal antibody raised against rat liver CYP1A1/1A2 isoenzymes to the microsomes isolated from control neuronal and glial cells did not produce any significant inhibition in the EROD activity at lower concentrations. Sig-
Fig. 3. a) Ethidium bromide stained agarose gel showing rat CYP1A2 mRNA in cultured neuronal and glial cells. Lane 1 contains 5 μl of the RT-PCR product without RNA. Lanes 2 and 3 contains 5 μl of the RT-PCR product isolated from control and 3-MC treated glial cells. Lanes 4 and 5 contain 5 μl of the RT-PCR product of RNA isolated from control and 3-MC treated neuronal cells. Lanes 6 and 7 contain 5 μl of the RT-PCR product isolated from control liver and 1.0 Kb DNA ladder respectively. b) Densitometric analysis of RT-PCR products (mean ± 3 gels). G and N correspond to the intensity of RT-PCR product of RNA isolated from cultured glial and neuronal cells respectively.
nificant inhibition in the microsomal EROD activity was observed only at the highest concentration of the antibody used (Table 2). Both, cultured neuronal or glial cells exhibited almost similar magnitude of inhibition in the enzyme activity on addition of the polyclonal anti-CYP1A1/1A2 to the reaction mixture (Table 2). Addition of preimmune IgG had no significant effect when added in vitro to the microsomes isolated from control neuronal or glial cells (Table 2). In vitro addition of antiCYP1A1/1A2 to the microsomes isolated from 3-MC pretreated neuronal or glial cells produced a significant concentration dependent inhibition of the enzyme activity at all the concentrations studied with approximately 90% inhibition of the enzyme activity occurring at the highest concentration of the antibody (Table 2). Reverse transcriptase-PCR analysis
Fig. 2. a) Ethidium bromide stained agarose gel showing rat CYP1A1 mRNA in cultured neuronal and glial cells. Lane 1 contains 5 μl of the RT-PCR product without RNA. Lanes 2 and 3 contain 5 μl of the RT-PCR product isolated from control and 3-MC treated glial cells respectively. Lanes 4 and 5 contain 5 μl of the RT-PCR product of RNA isolated from control and 3-MC treated neuronal cells respectively. Lanes 6 and 7 contain 5 μl of the RT-PCR product isolated from control liver and 1.0 Kb DNA ladder respectively. b) Densitometric analysis of RT-PCR products (mean ± 3 gels). G and N correspond to the intensity of RT-PCR product of RNA isolated from cultured glial and neuronal cells respectively.
Reverse transcriptase-PCR analysis with primers specific for rat liver GAPDH resulted in the formation of PCR products of expected band size of 194 bp in the RNA isolated from the rat brain neuronal and glial cells cultured in DMEM alone or in DMEM plus 3-MC (Fig. 1). Densitometric analysis of the PCR products revealed that PCR amplification resulted in the formation of products of almost equal intensity (Fig. 1). Using primers specific for CYP1A1 and CYP1A2, PCR analysis of the RT product obtained from the RNA isolated from rat brain neuronal and glial cells cultured in DMEM alone or in DMEM
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Fig. 4. Immunocytochemical detection of CYP1A1 in cultured rat brain neuronal cells. a, b and c represent primary cultures of neuronal cells in DMEM. d, e and f represent cultures of neuronal cells in DMEM + MC. a and d show cells in culture that are positive for β-III tubulin (red-TRITC), a neuronal marker. b and e show immunoreactivity in the same neuronal cells with anti-CYP1A1 (green-FITC). c and f represent an overlay of the two images control and MC treated respectively. Original magnification ×40, scale 20 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 5. Immunocytochemical detection of CYP1A1 in cultured rat brain glial cells. a, b and c represent primary cultures of glial cells in DMEM. d, e and f represent cultures of glial cells in DMEM + MC. a and d show cells in culture that are positive for anti-GFAP (red-TRITC), a glial marker. b and e show immunoreactivity in the same glial cells with anti-CYP1A1 (green-FITC). c and f represent an overlay of the two images control and MC treated respectively. Original magnification ×40, scale 20 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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plus 3-MC, after normalization with GAPDH produced a distinct band visible by agarose gel electrophoresis and of the correct size of 341 bp and 793 bp respectively (Figs. 2 and 3). PCR products of similar size were obtained from the RNA isolated from control rat liver, using the same primers. The basal expression of CYP1A1 and CYP1A2 mRNA was more in cultured neuronal cells as compared to the glial cells. Treatment of the cultured brain cells with MC produced a significant increase in CYP1A1 and CYP1A2 mRNA expression in both neuronal and glial cells, relative to the corresponding untreated cells. However, densitometric analysis revealed that the percentage of induction was more in glial cells as compared to the neuronal cells. CYP1A1 was found to be induced to a greater extent as compared to CYP1A2 (Figs. 2 and 3). Western blot analysis Immunoblot analysis of the solubilized microsomal proteins isolated from the untreated or treated neuronal and glial cells with hepatic anti-CYP1A1/2A2 did not exhibit detectable immunoreactivity co-migrating with the liver isoenzyme in either control rat brain cells or cells cultured in the presence of 3-MC (data not shown). Assessment of CYP1A1/1A2 expression in cultured neuronal and glial cells by immunocytochemistry Both neuronal and glial cells cultured with DMEM alone or treated with MC, grown on PLL coated glass slides, when fixed, and stained with anti-CYP1A1 antibody and with anti-β-III tubulin or anti-GFAP, and the secondary antibodies labeled with FITC or TRITC showed positive staining for CYP1A1. As shown in Fig. 4, all the neuronal cells exhibiting staining with anti-β-III tubulin also expressed CYP1A1 as judged by similar pattern of staining with anti-CYP1A1. Likewise, all glial cells exhibiting staining with anti-GFAP also expressed CYP1A as judged by staining with anti-CYP1A (Fig. 5). 3-MC treatment was not found to have any effect on the immunoreactivity observed with anti-GFAP or anti-β-III tubulin alone in either glial or neuronal cells (Figs. 4 and 5). As monoclonal antiGFAP used recognizes astrocytes and Bergman glial cells only, some of the cells immunopositive for CYP1A are negative for anti-GFAP, as it is a mixed glial culture from whole brain, which may also contain the oligodendrocytes. Similarly, some anti-βIII tubulin negative cells, which were immunopositive for CYP1A, could be the endothelial or fibroblast cells contaminating the neuronal cultures. Software analysis (Leica Qfluro Standard, Leica Microsystems Imaging Solutions Ltd., Version V1.2.0) revealed that treatment of 3-MC resulted in an increase in the intensity of FITC fluorescence. The mean intensity of FITC in treated neuronal cells was increased (27%) as compared to the control cells (Fig. 4). Likewise the glial cells exhibited an increased (174%) intensity of fluorescence of treated cells when compared to the control cells (Fig. 5). Moreover, the percentage of induction was more in glial cells than that observed in neuronal cells.
Discussion Expression of CYP1A isoenzymes and their catalytic activity in cultured brain cells is consistent with the recent studies indicating the presence of arylhydrocarbon receptor (Ahr) system in primary cultures of rat neuronal and glial cells (Filbrandt et al., 2004; Pravettoni et al., 2005). Kainu et al. (1995) have also earlier reported the localization of the main components of the Ahr pathway in the neurons of several brain areas of adult male rats. Several fold increase in the EROD activity as well as the expression of CYP1A isoenzymes in the cultured brain cells following treatment of 3-MC have indicated that responsiveness of CYP1A isoenzymes to CYP inducers is retained in the primary cultures of brain cells. Though earlier studies have demonstrated that glial cells harvested from rat brain tissues respond to CYP1A1/1A2 inducers, not much information is available about the responsiveness of cultured neurons (Legare et al., 1997; Filbrandt et al., 2004). Interestingly, cultured neurons exhibit relatively higher activity of EROD than the glial cells, though the magnitude of induction in the enzyme activity after in vitro addition of 3-MC was less in the neurons when compared to the glial cells. Immunocytochemistry, using specific antibodies for CYP1A1/ 1A2 and RT-PCR, with primers specific for rat liver CYP1A1and CYP1A2, have also shown higher immunoreactivity and mRNA expression of CYP1A1 and CYP1A2 in cultured neurons than the glial cells. However, the inability to detect CYP1A1/1A2 protein expression by immunoblotting in either control or 3-MC treated cultured neuronal and glial cells could be because of the relatively lower protein expression of CYP1A1/1A2 in brain cells which maybe beyond the levels of detection of the antibody used in the present study. Filbrandt et al. (2004) have also reported that because of relatively lesser expression of CYP1A1 in astrocytes, CYP1A1 expression and inducibility is not detected at the protein level by immunoblotting. However, induction of CYP1A1 protein expression has been shown in rat C6 glial cells with TCDD treatment (Takanaga et al., 2001). This discrepancy could be attributed to the use of a glial cell line rather than the primary cultures of brain cells used in the present study or reported earlier (Filbrandt et al., 2004). In contrast, the ability to detect responsiveness of cultured brain cells to 3-MC by immunocytochemistry or PCR could be because of the enhanced sensitivity of these assays where even the increase in the expression of single cell could be detected as compared to identifying increase in protein expression from a pool of microsomal proteins by western blotting. Relatively higher activity of CYP1A1/1A2 in cultured neurons have further provided support to the hypothesis that xenobiotic metabolizing CYPs have a physiological role in brain (Hedlund et al., 2001; Nissbrandt et al., 2001; Strobel et al., 2001; Miksys and Tyndale, 2002; Parmar et al., 2003; Gervasini et al., 2004). Konstandi et al. (2005), using selected agonists and antagonists have identified the role of α and β-adrenoreceptors in the regulation of CYP1A1 induction in brain. Adrenoreceptors were found to be involved in the regulation of the CYP1A1 gene at mRNA level. Both, reduced noradrenaline release in CNS and central catecholamine depletion
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resulted in downregulation of CYP1A1 expression (Konstandi et al., 2004, 2005). Recent studies have indicated a role of CYPs in neurotransmission and possible endogenous substrates for CYP2E1, CYP1A2 and CYP2D6 have been identified in the brain. A role of CYP1A isoenzymes in the hydroxylation of estradiol, which is involved in dopamine receptor supersenstivity, has also been demonstrated (Tsuchiya et al., 2005). Although the exact role of CYPs in human brain needs to be elucidated, modulation of CYP1A2 microsomal enzyme activity by serotonin and tryptamine, the two indolamines have lead to the speculation that neurotransmitters and other substances whose local concentrations are finely tuned by several mechanisms, might play a regulatory role on brain CYP1A2 activity (Agundez et al., 1998; Martinez et al., 2000). This local regulatory role of endogenous substances might also possibly help in explaining the relatively higher degree of induction in enzyme activity (EROD) in brain cells, as observed in the present study after the addition of MC, rather than the protein expression. Poor correlation in liver samples between CYP1A2 content, as measured by western immunoblotting and CYP1A2 activity as measured with specific substrates has been reported in the liver samples of patients undergoing surgery (Lucas et al., 1993). This lack of correlation was not attributed to smoking habits or alcohol intake but rather to the influence of local factors or possible endogenous substrates that modulate enzyme activity rather than enzyme expression. Interestingly, relatively higher magnitude of induction of CYP1A isoenzymes in cultured glial cells could be of significance as these cells are the main cellular components of the blood–brain barrier (BBB). As glial cells have an important physiological role in integrating neuronal inputs, neurotransmitter release and the protection and repair of nervous tissue, the greater responsiveness of CYP1A isoenzymes to CYP1Ainducers in glial cells could be attributed to their role in toxication–detoxication mechanisms. The greater magnitude of induction of CYP1A isoenzymes in glial cells has further provided support to the assumption that glial cells function as a control element for the xenobiotics, which reach the blood– brain barrier. Earlier studies have shown that stimulation of astrocytes, the major glial cells in the CNS, with mediators of inflammation results in downregulation of CYP1A activity in cultured astrocytes (Nicholson and Renton, 1999, 2002; Abdulla and Renton, 2005). Recent evidences have shown that several CYP isoforms including CYP1A found in the brain are depressed during a localized CNS inflammatory response (Nicholson and Renton, 2001, 2002). However, β-adrenoreceptor stimulation during conditions of CNS inflammation prevented the downregulation in CYP1A1 and CYP1A2 activity in astrocytes, demonstrating the protective role of β-adrenoreceptors during CNS inflammation (Abdulla and Renton, 2005). In conclusion, the present study clearly demonstrates the expression of CYP1A1 and CYP1A2 in both the cultured neuronal and glial cells. Higher levels of these isoforms in cultured brain cells support the physiological role of CYP1A in brain. The greater magnitude of induction of CYP1A in glial cells is of significance, as these cells are components of the
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blood–brain barrier and it is suggested that they have a potential role in the toxication–detoxication mechanism. However, induction of CYP1A in neurons could be of toxicological significance, as alterations in the levels of this CYP isoforms could also result in modulating the physiological effects regulated by these isoenzymes in brain. Acknowledgement Authors are grateful to Director, ITRC for his keen interest in the work. The financial assistance of the Council of Science & Technology, U.P and CSIR, India for carrying out the above studies is gratefully acknowledged. The technical assistance of Mr. B. S. Pandey and Mr. Rajesh Misra and computer help of Mr. Mohd. Aslam is gratefully acknowledged. ITRC Publication No.: 2482. References Abdulla, D., Renton, K.W., 2005. Beta-adrenergic receptor modulation of the LPS-mediated depression in CYP1A activity in astrocytes. Biochemical Pharmacology 69, 741–750. Agundez, J.A., Gallardo, L., Martinez, C., Gervasini, G., Benitez, J., 1998. Modulation of CYP1A2 enzyme activity by indoleamines: inhibition by serotonin and tryptamine. Pharmacogenetics 8, 251–258. Anandatheerthavarada, H.K., Shankar, S.K., Ravindranath, V., 1990. Rat brain cytochromes P-450: catalytic, immunochemical properties and inducibility of multiple forms: Preparation of brain microsomes with cytochrome P450 activity using calcium aggregation method NADPH cytochrome P-450 reductase in rat, mouse and human brain. Brain Research 536, 339–343. Cherng, S.H., Lin, P., Yang, J.L., Hsu, S.L., Lee, H., 2001. Benzo[g,h,i]perylene synergistically transactivates benzo[a]pyrene-induced CYP1A1 gene expression by aryl hydrocarbon receptor pathway. Toxicology and Applied Pharmacology 170, 63–68. Eaton, D.L., Gallagher, E.P., Bammler, T.K., Kunze, K.L., 1995. Role of cytochrome P450 1A2 in chemical carcinogenesis: implications for human variability in expression and enzyme activity. Pharmacogenetics 5, 259–274. Filbrandt, C.R., Wu, Z., Zlokovic, B., Opanashuk, L., Gasiewicz, T.A., 2004. Presence and functional activity of the aryl hydrocarbon receptor in isolated murine cerebral vascular endothelial cells and astrocytes. Neurotoxicology 25, 605–616. Gervasini, G., Carrillo, J.A., Benitez, J., 2004. Potential role of cerebral cytochrome P450 in clinical pharmacokinetics: modulation by foreign chemicals. Clinical Pharmacokinetics 43, 693–706. Gonzalez, F.J., Matsunaga, T., Nagata, K., 1989. Structure and regulation of P450s in the rat P450IIA gene subfamily. Drug Metabolism Reviews 20, 827–837. Guengerich, F.P., 1990. Characterization of roles of human cytochrome P450 enzymes in carcinogen metabolism. Asia Pacific Journal of Pharmacology 5, 327–345. Hedlund, E., Gustafsson, J.A., Warner, M., 2001. Cytochrome P450 in the brain; a review. Current Drug Metabolism 2, 245–263. Hodgson, A.V., White, T.B., White, J.W., Strobel, H.W., 1993. Expression analysis of the mixed function oxidase system in rat brain by the polymerase chain reaction. Molecular and Cellular Biochemistry 120, 171–179. Johri, A., Dhawan, A., Singh, R.L., Parmar, D., 2006. Effect of prenatal exposure of deltamethrin on the ontogeny of xenobiotics metabolizing cytochrome P450s in the brain and liver of offsprings. Toxicology and Applied Pharmacology 214, 279–289. Kainu, T., Gustafsson, J.A., Pelto-Huikko, M., 1995. The dioxin receptor and its nuclear translocator (Arnt) in the rat brain. Neuroreport 6, 2557–2560. Kapitulnik, J., Gelboin, H.V., Guengerich, F.P., Jacobowitz, D.M., 1987. Immunohistochemical localization of cytochrome P-450 in rat brain. Neuroscience 20, 829–833.
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