Evidence for cytochrome P450 2E1 catalytic activity and expression in rat blood lymphocytes

Life Sciences 77 (2005) 1082 – 1093 www.elsevier.com/locate/lifescie

Evidence for cytochrome P450 2E1 catalytic activity and expression in rat blood lymphocytes Aparajita Dey1, Alok Dhawan, Prahlad Kishore Seth, Devendra ParmarT Developmental Toxicology Division, Industrial Toxicology Research Centre, P.O. Box 80, M.G. Marg, Lucknow-226 001, U.P., India Received 12 July 2004; accepted 4 January 2005

Abstract Studies initiated to characterize cytochrome P450 2E1(CYP2E1) in freshly isolated rat blood lymphocytes revealed significant mRNA of CYP2E1 in control blood lymphocytes. RT-PCR studies have shown that as observed in liver, acute treatment of ethanol (single oral dose of 0.8 ml/kg b.wt , i.p), resulted in increase in the mRNA expression of CYP2E1 in freshly isolated rat blood lymphocytes. Western blotting studies using polyclonal antibody raised against rat liver CYP2E1 demonstrated significant immunoreactivity, comigrating with the liver isoenzyme, in freshly isolated control rat blood lymphocytes. Similar to that seen in liver, pretreatment of ethanol was found to produce an increase in the CYP2E1 isoenzyme in the blood lymphocytes. Blood lymphocytes were also found to catalyze the CYP dependent N-demethylation of N-nitrosodimethylamine (NDMA), which like in liver increased 2–3 fold following pretreatment of rats with known CYP2E1 inducers. Kinetic studies have further shown significant increase in the apparent Vmax and the affinity towards the substrate in rat blood lymphocytes indicating that as observed in liver, the increase in mRNA and protein expression following exposure to CYP2E1 inducers is associated with the increased catalytic activity of CYP2E1 in freshly isolated rat blood lymphocytes. The data indicating similarities of the blood lymphocyte CYP2E1 with the liver enzyme suggest that lymphocyte CYP2E1 levels in freshly isolated rat blood lymphocytes could be used to monitor tissue enzyme levels. D 2005 Elsevier Inc. All rights reserved. Keywords: Cytochrome P450 2E1; Blood; Liver; RT-PCR; Western blotting; Enzyme

T Corresponding author. Fax: +91 522 2628227, 2211547, 2227390. E-mail address: [email protected] (D. Parmar). 1 Present address: Department of Pharmacology and Biological Chemistry, Mt. Sinai School of Medicine, One Gustave L. Levy Place, Box 1603, Annenberg Building, Room 19–20, New York, NY 10029, U.S.A. 0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2005.01.021

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Introduction Cytochrome P450 2E1 (CYP2E1) plays an important role in determining the toxicity of numerous environmental chemicals due to its capacity to metabolize solvents and other environmental procarcinogens to their cytotoxic and / or carcinogenic metabolites (Koop, 1992; Lieber, 1991). Xenobiotics such as ethanol, acetone, pyridine, pyrazole, isoniazid and pathophysiological conditions such as diabetes, fasting, obesity, high fat diet and long term alcohol consumption have been found to induce the expression of CYP2E1 in experimental animals and humans resulting in significantly enhanced toxicity (Raucy, 1995). CYP2E1 has also been shown to be involved in the increased incidence of liver disease and cancer in diabetics, alcoholics and obese individuals (Andersen et al., 1984; Koop, 1992; Lieber et al., 1979; Lieber, 1988, 1991; Raucy, 1995). Polymorphisms for the restriction enzymes Rsa 1 and Pst 1 have been detected in the 5’-upstream sequence of the gene encoding the human CYP2E1. These polymorphisms exhibit interethnic variation and may determine differences amongst the individuals towards the toxicity induced by xenobiotics (Boobis, 1992). Monitoring of CYP2E1 levels in individuals exposed to environmental agents, known to be inducers of CYP2E1 or pathophysiologic conditions resulting in induction of CYP2E1 could help in the identification of individuals who may be at an increased risk. The use of metabolic markers like 6hydroxylation of chlorzoxazone or N-1 and N-7 demethylation of caffeine may be used to assess in vivo CYP2E1 expression but these methods have disadvantages since these probes may not be solely metabolized by CYP2E1. Under certain circumstances or induction of other CYPs may occur which may contribute significantly to the reaction that is being monitored (Raucy, 1995; Streetman et al., 2000). Furthermore, the use of metabolic probes requires multiple sampling of urine and blood and an extended assessment period, usually in a hospital setting. Instead of using metabolic markers to assess CYP2E1 in vivo levels, another alternative could be to use blood lymphocytes to monitor its expression provided CYP2E1 levels in lymphocytes parallel those in liver. CYP2E1 has been shown to be expressed in blood lymphocytes (Raucy et al., 1995, 1997; Scobbie and Mason, 1999; Soh et al., 1996; Song et al., 1990). However, most of these studies have been performed in microsomes isolated from cultured blood lymphocytes and which require addition of mitogens to stimulate CYP enzyme activities. Recent studies from our laboratory have shown that freshly isolated intact peripheral blood lymphocytes could be used as a tool to estimate CYP levels (Dey et al., 2001, 2002). These studies reported similarities in the expression and regulation of lymphocyte CYP1A1 with the liver isoenzyme (Dey et al., 2001). Freshly isolated intact rat blood lymphocytes were also found to catalyze CYP2E1 dependant lipid peroxidation and demethylation of N-nitrosodimethylamine, NDMA (Dey et al., 2002). To further investigate if the freshly isolated peripheral blood lymphocytes CYP2E1 could be used as a rapid screen to monitor hepatic changes, studies were initiated to investigate the similarities in the expression and regulation of CYP2E1 in blood lymphocytes with the liver isoenzyme. Materials and methods Chemicals N-nitrosodimethylamine (NDMA), 3-methylcholanthrene (MC), phenylmethyl sulfonyl fluoride (PMSF), NADPH, bovine serum albumin (BSA), Histopaque 1077 and dithiothreitol (DTT) were

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procured from Sigma-Aldrich, St. Louis, MI, U.S.A. Ethanol was obtained from Bengal Chemicals Ltd. All the other chemicals used were of highest purity commercially available and were procured either from E. Merck, India or Sisco Research Laboratories Pvt. Ltd., India. Phenobarbitone sodium salt (PB) was a gift from Biodeal Laboratories, India. Antiserum for CYP2E1 was procured from Oxygene, Dallas, USA. Immobilon P-membrane was procured from Millipore Corp., USA. Animals and treatment Adult male albino Wistar rats (~ 8 week old) were procured from Industrial Toxicology Research Centre breeding colony and raised on Amrut animal feed (now Maharashtra Chakan Oil Mill Ltd., Pune, Maharashtra, India) and water ad libitum. Animal care and experimentation was in accordance with the policy laid down and was approved by the Animal Care Committee of the Centre. The animals were divided into five groups of ten animals each. Rats in the first and second group received a single dose of ethanol (0.8 ml/kg body weight, i.p.) or pyrazole (200 mg/kg b. wt., i.p.) for four consecutive days respectively. The animals in the third group received phenobarbital (PB, 80 mg/kg, b.wt.) dissolved in corn oil daily intraperitoneally (i.p.) for five consecutive days while animals in the fourth group received 3-methylchloanthrene (MC, 30 mg/kg b.wt.), dissolved in corn oil for five consecutive days. The animals in the fifth group served as the control. Blood was drawn from the choroid plexus of the animals 16 hrs after the last dose. Isolation of lymphocytes Lymphocytes were isolated from the blood by the method of Boyum (Boyum, 1968) with slight modifications. In brief, 4.0 ml of whole blood was diluted with 4.0 ml of phosphate buffered saline (PBS), pH 7.4, and carefully layered over 2.0 ml of Histopaque 1077. After centrifugation at 400 x g for 30 min at room temperature, the upper layer was discarded and the opaque interface containing mononuclear cells was transferred into a clean centrifuge tube. After repeated washing of the lymphocytes with PBS and recentrifugation at 250 x g, the resulting pellet was resuspended in 0.5 ml of PBS and used for enzyme estimations and immunoblotting. The number of lymphocytes was counted using a haemocytometer and the viability of the cells was assayed by the trypan blue exclusion test. Enzyme assay NDMA-d activity was assayed by a slight modification of the method of Castonguay et al., (Castonguay et al., 1991). The assay mixture contained a suitable amount of lymphocytes, 70.0 mM Tris-HCl, pH 7.4, 10.0 mM semicarbazide, 14.0 mM MgCl2, 215.0 mM KCl, 1.0 mM NADPH and 4.0 mM NDMA in 1.0 ml final volume. The reaction mixture was incubated at 37 8C for 30 minutes and the reaction was stopped by the addition of 0.1 ml of 25% zinc sulphate and 0.1 ml of saturated solution of barium hydroxide. After centrifugation at 2000 rpm for 10 minutes, 0.7 ml of the supernatant was mixed with an equal amount of Nash reagent. The tubes were then incubated at 70 8C for 20 min and the HCHO formed was measured at 415 nm (Nash, 1953). The apparent Km and Vmax was determined using a total of 10–14 substrate concentrations over the range of 2.0 mM to 200.0 mM in control rats and 2.0 mM to 32.0 mM in ethanol pretreated rats. These values were based

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on the Km and Vmax values obtained for the activity of NDMA-d in microsomes isolated from livers of control and ethanol pretreated rats (Peng et al., 1982). Protein content of the samples was estimated by the method of Lowry et al., (Lowry et al., 1951) using bovine serum albumin as the reference standard. Statistical analysis Student’s tV test was employed to calculate the statistical significance between control and treated groups. p b 0.05 was considered to be significant when compared with the controls. Immunoblot analysis Prior to immunoblotting studies lymphocytes were sonicated in ice and then centrifuged at 10,000 rpm for 20 minutes. The resulting supernatant was solubilised in buffer containing 1.0 mM dithiothreitol, 1.0 mM EDTA, 0.2% emulgen 911 and 20% glycerol for one hour at 4 8C. After solubilisation, samples were recentrifuged at 10,000 rpm for 20 minutes and used for immunoblotting studies. The lymphocyte supernatant was subjected to SDS-PAGE (3% acrylamide stacking gel and 7.5% acrylamide separating gel) and processed for western blotting according to the method of Towbin et al., (Towbin et al., 1979) as modified by Parmar et al., (Parmar et al., 1998). The blots were scanned in VERSA DOC Imaging system, Model 1000 (Biorad, USA) and densitometric analysis of the bands was carried out using Quantity One Quantitation Software Version 4.3.1 (Biorad, U.S.A). RNA isolation Total RNA was extracted from whole blood isolated from control and ethanol pretreated rats by TRIzol BD and from liver by TRIzol reagent (Life Technologies, U.S.A) according to the manufacturer’s protocol. The protocol utilizing TRIzol reagent, a monophasic solution of phenol and guanidium isothiocyanate, is an improvement to the single-step RNA isolation developed by Chomczynski and Sacchi (Chomczynski and Sacchi, 1987). RT-PCR analysis cDNA was synthesised by first denaturing reaction mixture containing 5 Ag RNA, 5 AM Oligo(dT)20 and DEPC treated autoclaved water at 65 8C for 5 min and subsequently incubating at 4 8C. For reverse transcription, the reaction mixture in 10 Al contained 1X cDNA synthesis buffer, 0.01 M DTT, 4U/Al RNAse Out, 2 mM dNTP mix and Thermoscript RT (1.5 units /Al) and reaction was carried out in a Stratagene Robocycler by incubating the above at 50 8C for 60 min. The reaction was terminated by incubating the mixture at 85 8C for 5 min. RNAse H (1 Al) was then added to the cDNA and the mixture was incubated at 37 8C for 20 min. 2 Al (10%) of the cDNA formed was used for subsequent PCR reactions. The reaction mixture for PCR of CYP2E1 in 50 Al contained 1X High Fidelity PCR buffer, 2.0 mM MgSO4, 0.2 mM dNTP mix, 0.2 AM of each CYP2E1 primers, 2 Al of cDNA and 0.2 Al Taq High Fidelity enzyme. The sequence of primers used for PCR reactions were specifically designed for CYP2E1, the details of which were described by Soh et al., (Soh et al., 1996). PCR was carried out in Stratagene Robocycler using 35 cycles of denaturation at 94 8C for 1

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min, annealing of primers at 50 8C for 30 sec, extension at 72 8C for 1min and final extension at 72 8C for 5 min. 2 Al of the product of the 1st PCR was reamplified using nested primers for CYP2E1 following the same conditions. PCR was also carried out for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a housekeeping gene. The reaction mixture for PCR in 50 Al contained 1X High Fidelity PCR buffer, 2.0 mM MgSO4, 0.2mM dNTP mix, 0.2 AM of each GAPDH primers (Soh et al., 1996), 2 Al of cDNA and 0.2 Al Taq High Fidelity enzyme. PCR was carried out using 35 cycles of denaturation at 94 8C for 1 min, annealing of primers at 55 8C for 1 min, extension at 72 8C for 1min and final extension at 72 8C for 10 min. PCR products (750bp for CYP2E1 and 194bp for GAPDH) were analysed by electrophoresis in 1% and 1.8% agarose gel respectively stained with ethidium bromide 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). 1

A

2

3

4

5

6

1.0

0.5

B 400 Control

Densitometric units (DU)

350

Ethanol

300 250 200 150 100 50 0 L

Ly

Fig. 1. A-Ethidium bromide stained agarose gel showing rat CYP2E1 mRNA in blood and liver. Lane 1 contains 1.2 Kb DNA ladder. Lane 2 contains 5 Al of the RT-PCR product without RNA. Lanes 3 and 4 contain 5 Al of the RT-PCR product isolated from blood of ethanol treated and control rats respectively. Lanes 5 and 6 contain 5 Al of the RT-PCR product of RNA isolated from liver of ethanol pretreated and control rats respectively; B- Densitometric analysis of RT-PCR products. L corresponds to the intensity of RT-PCR product of RNA isolated from liver. Ly corresponds to the intensity of RT-PCR product of RNA isolated from blood lymphocytes.

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Results RT-PCR analysis Results of RT-PCR amplification of RNA, isolated from the liver or whole blood of control rats or rats pretreated with ethanol, with primers specific for hepatic CYP2E1 and GAPDH, are shown in Figs. 1A and B and 2A and B respectively. As evident from Fig. 1A, PCR product of the expected band size of 750 bp was formed with the RNA isolated from the liver of the control rats. Similar product was obtained from the RNA isolated from the liver of ethanol-pretreated rats with slight induction being observed in the product formed after ethanol pretreatment (Figs. 1A and B). RT-PCR amplification of the RNA isolated from the blood lymphocytes of control or ethanol pretreated rats also produced a PCR product of the same size as of the liver. An increase in the intensity of the band, almost similar to that seen in the liver, was observed in the lymphocytes isolated from ethanol pretreated rats when compared with that of RNA obtained from blood lymphocytes of control rats (Figs. 1A and B). RT-PCR analysis with primers specific for rat liver GAPDH resulted in the formation of PCR products of expected band size of 194bp in the RNA isolated from the liver or A

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200 100

Control

Ethanol

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Fig. 2. A-Ethidium bromide stained agarose gel showing rat GAPDH mRNA in blood and liver. Lane 1 contains 1 Kb DNA ladder. Lane 2 contains 5 Al of the RT-PCR product without RNA. Lanes 3 and 4 contain 5 Al of the RT-PCR product isolated from blood of ethanol treated and control rats respectively. Lanes 5 and 6 contain 5 Al of the RT-PCR product of RNA isolated from liver of ethanol pretreated and control rats respectively. B- Densitometric analysis of RT-PCR products. L corresponds to the intensity of RT-PCR product of RNA isolated from liver. Ly corresponds to the intensity of RT-PCR product of RNA isolated from blood lymphocytes.

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blood lymphocytes of control rats or rats pretreated with ethanol (Fig. 2A). Furthermore, densitometric analysis of the PCR products revealed almost similar intensity of the bands formed in liver or blood lymphocytes of control or ethanol pretreated rats (Fig. 2B). Western blot analysis A significant crossreactivity of polyclonal antibody raised against rat liver CYP2E1 was observed with blood lymphocytes isolated from control rats. The immunoreactivity observed in control blood lymphocytes was found to comigrate with the liver isoenzyme (Fig. 3A). Based on the migration of the standard and on the Rfs, immunoreactivity observed in the blood lymphocytes in the molecular weight range of 52 kDa was identified as CYP2E1. An increase in the immunoreactivity was observed with antiCYP2E1 with increasing concentration of blood lymphocyte protein. Pretreatment of rats with ethanol was found to increase the expression of CYP2E1 in the blood lymphocytes as reflected by increased immunoreactivity when compared with that observed with control blood lymphocytes (Figs. 3A and B). Enzymatic studies Significant NDMA-d activity was found in rat blood lymphocytes, which was essentially dependent on NADPH (Table 1). When NADPH was substituted with NADH, no enzyme activity could be A

Densitometric units (DU)

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300 Control

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Ethanol 200 150 100 50 0

L (25µg)

Ly (25µg)

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Fig. 3. Western blot analysis of rat lymphocyte protein with anti-P450 2E1. (Lane 1, 25Ag microsomal protein from liver of ethanol pretreated rats; lanes 2, 4 and 6: 150, 100 and 75 Ag lymphocyte protein from ethanol pretreated rats respectively; lanes 3, 5 and 7: 150, 100 and 75 Ag lymphocyte protein from control rats respectively. Arrow indicates the mobility of the standard of M.W. 55.4 kDa). B Densitometric analysis of immunoblot. L corresponds to intensity of band corresponding to CYP2E1 of liver of ethanol treated rat. Ly corresponds to intensity of bands corresponding to CYP2E1 in blood lymphocytes. Data in parenthesis indicate the amount of protein loaded in the gel.

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Table 1 Cofactor requirement for NDMA-d a activity in rat blood lymphocytes Conditions

Blood lymphocyte

Liver

Complete Complete Complete Complete Complete

1.01 F 0.2 1.02 F 0.4 N.D. 0.56 F 0.1T N.D.

2.68 F 0.2 2.75 F 0.3 b 0.2 1.60 F 0.2T b 0.2

system system+NADH system-NADPH system+SKF 525A system+CO

Complete system represents 70.0 mM Tris-HCl, buffer, pH 7.4 + 10 mM semicarbazide + 1.0 mM NADPH + 4.0 mM NDMA +14.0 mM MgCl2 + 215.0 mM KCl. SKF 525-A was added at the final conc. of 0.001 M. All values are mean F S.E. of 3 experiments. N.D. = not detected. a nmoles HCHO formed/min/mg protein. T p b 0.05 when compared to the controls.

detected in the lymphocytes. Similarly, no significant change in the enzyme activity was observed when NADH (1.0 mM) was added to the reaction mixture containing NADPH (1.0 mM). Addition of SKF 525-A (1.0 mM), an inhibitor of CYP catalysed reactions, to the reaction mixture resulted in significant inhibition of NDMA-d activity. Likewise, no activity of NDMA-d was detected in lymphocytes when the reaction mixture was saturated with carbon monoxide prior to the addition of substrate (Table 1). Pretreatment of rats with ethanol or pyrazole or acetone, selective inducers of CYP2E1, produced an significant increase in the activity of NDMA-d in rat blood lymphocytes. Similar increase in the enzyme activity following exposure of these different inducers was observed in liver microsomes, though the magnitude of induction was more when compared with intact blood lymphocytes (Table 2). Pretreatment of rats with 3-methylcholanthrene (MC), CYP1A1/1A2 inducer failed to produce any significant change in the activity of NDMA-d in either liver microsomes or freshly isolated rat blood lymphocytes. No significant increase in the activity of NDMA-d was observed in rat blood lymphocytes isolated from rats pretreated with PB, an inducer of CYP2B1/2B2 catalyzed reactions, as compared to small but significant increase in the enzyme activity in liver microsomes (Table 2). A monophasic pattern of kinetics was observed with NDMA-d enzyme in blood lymphocytes isolated form both control and ethanol pretreated rats (Table3). Line weaver Burke plot revealed that ethanol Table 2 Effect of various inducers on NDMA-d a activity in blood lymphocytes Control Pyrazole Acetone Ethanol PB MC All the values are mean F S.E. of five animals. a nmoles HCHO formed/min/mg protein. T p b 0.05 when compared to the controls.

Blood lymphocyte

Liver

1.06 F 0.3 2.33 F 0.1T 2.18 F 0.2T 2.71 F 0.6T 1.15 F 0.4 0.82 F 0.3

2.68 F 0.2 9.10 F 1.3T 8.85 F 1.2T 9.40 F 1.3T 4.05 F 1.1T 2.50 F 0.7

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Table 3 Apparent kinetic constants for rat lymphocyte NDMA-d Control Ethanol

Kma

Vmaxb

2.1 F 0.3 1.3 F 0.1T

1.2 F 0.4 2.5 F 0.2T

Values represent data F S.E. of three experiments. a Km = mM. b Vmax = HCHO formed /min/ mg protein. T p b 0.05 when compared to the controls.

pretreatment resulted in significant increase in the affinity of the enzyme towards the substrate (apparent Km) when compared with control blood lymphocytes. Pretreatment of ethanol was also found to significantly increase the rate of reaction (apparent Vmax) when compared with lymphocytes isolated from control rats (Table 3).

Discussion Lymphocytes have obvious advantages in being used to develop non-invasive bioassays to screen human population for exposure to the toxicants. Though most of the studies in lymphocytes have been carried out in cultured lymphocytes in the presence of mitogens, recent studies have shown that freshly isolated peripheral blood lymphocytes express measurable levels of CYP enzymes (Dey et al., 2001, 2002; Fung et al., 1999; Raucy et al., 1999). The presence of their mRNA suggest the possibility of using blood lymphocytes as an indicator to study the status of CYP enzymes even though the role of these enzymes in blood lymphocytes is not fully understood (Baron et al., 1998; Dey et al., 2001; Fung et al., 1999). Recent studies from our laboratory have also indicated similarities in the regulation of lymphocytes CYPs with the liver enzyme (Dey et al., 2001, 2002). Consistent with the earlier reports indicating functional activity of CYP2E1 in rat blood lymphocytes (Dey et al., 2002), the present study have demonstrated significant mRNA expression of CYP2E1 in freshly isolated blood lymphocytes obtained from control rats. Soh et al., (Soh et al., 1996) earlier failed to detect constitutive mRNA expression of CYP2E1 in cultured blood lymphocytes. They attributed it to the extremely low concentration of the CYP2E1 mRNA expressed in uninduced blood lymphocytes. Furthermore an induction of CYP2E1 mRNA in lymphocytes isolated from ethanol-pretreated rats, have indicated increase in blood lymphocyte CYP2E1 at the pretranslational level (Raucy, 1995; Takahashi et al., 1993). This pretranslational induction of lymphocyte CYP2E1 has also been demonstrated earlier using a sensitive RNase protection assay in both the lymphocytes and liver (Soh et al., 1996). Since CYP2E1 mRNA contents do not always correspond to the protein levels (Elliasson et al., 1998; Roberts et al., 1995), western blotting studies were also performed to detect CYP2E1 in blood lymphocytes. Significant immunoreactivity of blood lymphocyte protein with rat hepatic anti-CYP2E1 has further provided evidence for the expression of CYP2E1 in blood lymphocytes. Similar pattern of expression of CYP2E1 protein was also reported earlier (Raucy, 1995; Raucy et al., 1999; Scobbie and Mason, 1999; Soh et al., 1996; Song et al., 1990). However, as compared to similar comigration of the lymphocyte CYP2E1 with the liver isoenzyme (~52KDa) observed in the present study, CYP2E1 was

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reported to be of low molecular weight (~ 46–48 KDa) and was attributed to the loading effect. Nevertheless, significant immunoreactivity observed in freshly isolated control blood lymphocytes and increase in CYP2E1 protein after pretreatment with ethanol is consistent with the RT-PCR data demonstrating similarities in the constitutive and inducible expression of CYP2E1 in blood lymphocytes with the liver isoenzyme. As observed by us, Soh et al., (Soh et al., 1996) have also provided immunochemical evidence for CYP2E1 and its induction in the homogenate and S9 fraction of the cultured blood lymphocytes from control and fasted rats. Song et al. (Song et al., 1990) reported extremely low or negligible levels of CYP2E1 in cultured lymphocytes isolated from humans while significantly elevated levels in lymphocytes isolated from poorly controlled insulin dependent diabetic patients. Though the cultured blood lymphocytes differ from freshly isolated lymphocytes, the apparent induction of CYP2E1 in the freshly isolated rat blood lymphocytes following ethanol pretreatment appears to be comparable to that observed with cultured blood lymphocytes isolated from fasted rats (Soh et al., 1996; Raucy et al., 1999). Our recent data indicating significant activity of CYP2E1 dependent NDMA-dand NADPHsupported lipid peroxidation in freshly isolated rat blood lymphocytes have further indicated that CYP2E1 expressed in blood lymphocytes is functionally and catalytically active (Dey et al., 2002). The inability of PB or MC or dexamethasone to significantly induce NDMA-d activity or NADPHdependent lipid peroxidation while significant (2 fold) induction observed with ethanol and other inducers have indicated that CYP2E1 expressed in blood lymphocytes catalyze the enzyme activity and NADPH dependent lipid peroxidation. Using in vitro metabolism of chlorzoxazone, a good correlation has also been earlier reported between CYP2E1 content in lymphocytes and blood alcohol concentrations. A greater chlorzoxazone clearance and lower area under the concentration curve was observed in blood lymphocytes of alcohol abusers than in non-alcoholic individuals (Raucy et al., 1997, 1999). Animal studies with rabbits ingesting 10–15% ethanol for different periods of time revealed higher hepatic and blood CYP2E1 levels when compared to animals receiving unsupplemented water (Raucy, 1995; Raucy et al., 1999). Further evidence that the increased mRNA and protein expression of CYP2E1 in rat blood lymphocytes was associated with the increased catalytic activity in blood lymphocytes was provided by the kinetic studies. Similar to that observed in liver, exposure to ethanol was found to significantly increase the rate of velocity of reaction associated with the significant increase in the affinity of the substrate towards the enzyme in peripheral blood lymphocytes. However, a relatively higher Km observed in the blood lymphocytes when compared to liver microsomes could be attributed to lower levels of CYP2E1 in the blood lymphocytes. As whole and intact blood lymphocytes were used as compared to the microsomal preparations of liver, it is likely that the other factors present in blood lymphocytes may have affected the activity of NDMA-d. Interestingly, a monophasic pattern of enzyme kinetics was observed in the blood lymphocytes isolated from control or ethanol pretreated rats. This appears to be slightly in contrast with the kinetics reported in control liver microsomes, where NDMA-d is demethylated by a multistep process in which CYP2E1 is only involved in the demethylation of high affinity form of NDMA-d while isoenzymes other than CYP2E1 are active at other substrate concentrations. However, similarities in the enzyme kinetics were observed in the blood lymphocytes and liver microsomes isolated from ethanol pretreated rats. The monophasic pattern of enzyme kinetics reported in liver microsomes after treatment of ethanol has been attributed to the selective enrichment of the high affinity (Km) form of NDMA-d in liver microsomes (Peng et al., 1982). These similarities and differences in the enzyme kinetics observed in blood lymphocytes suggests that unlike in liver

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microsomes, a single NDMA-d enzyme may exist in blood lymphocytes that is active at both high and low substrate concentrations (Lai and Arcos, 1980) and the levels of which are induced following exposure to the CYP2E1 inducers. To summarize, the results of the present study have clearly shown that CYP2E1 is expressed in rat blood lymphocytes and is functionally and catalytically active. Similarities in the expression and regulation of CYP2E1 in freshly isolated rat blood lymphocytes with the liver isoenzyme have provided evidence that freshly isolated intact blood lymphocytes could be used as a surrogate to monitor tissue CYP2E1 levels. Furthermore estimation of CYP2E1 in freshly isolated peripheral blood lymphocytes suggest that it can be used as a valuable and rapid biomarker to monitor CYP2E1 levels in the individuals who could be at risk to ethanol induced hepatotoxicity.

Acknowledgements Authors are grateful to Director, ITRC for his keen interest and support. AD is grateful to CSIR, New Delhi for awarding the Senior Research Fellowship. The technical assistance of Mr. B.S. Pandey and Mr. Rajesh Misra are gratefully acknowledged. ITRC Publication No. 2313.

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