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Cytochrome P450 2E1 dependent catalytic activity and lipid peroxidation in rat blood lymphocytes
Life Sciences 71 (2002) 2509 – 2519 www.elsevier.com/locate/lifescie
Cytochrome P450 2E1 dependent catalytic activity and lipid peroxidation in rat blood lymphocytes A. Dey a, D. Parmar a,*, A. Dhawan a, D. Dash b, P.K. Seth a a
Developmental Toxicology Division, Industrial Toxicology Research Centre, P.O. Box 80, M.G. Marg, Lucknow 226 001, India b Department of Biochemistry, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221 005, India Received 23 April 2001; received in revised form 12 October 2001; accepted 31 May 2002
Abstract To investigate the similarities in the catalytic activity of blood lymphocyte P450 2E1 in blood lymphocyte with the liver isoenzyme, NADPH dependent lipid peroxidation and activity of N-nitrosodimethyamine demethylase (NDMA-d) was studied in rat blood lymphocytes. Blood lymphocytes were found to catalyse NADPH dependent (basal) lipid peroxidation and demethylation of N-nitrosodimethylamine (NDMA). Pretreatment with ethanol or pyrazole or acetone resulted in significant increase in the NADPH dependent lipid peroxidation and the activity of NDMA-d in blood lymphocytes and liver microsomes. In vitro addition of CCl4 to the blood lymphocytes isolated from control or ethanol pretreated rats resulted in an increase in the NADPH dependent lipid peroxidation. Significant inhibition of the basal and CCl4 supported NADPH dependent lipid peroxidation and NDMA-d activity in blood lymphocytes isolated from control or ethanol pretreated rats by dimethyl formamide or dimethyl sulfoxide or hexane, solvents known to inhibit P450 2E1 catalysed reactions in liver and anti- P450 2E1, have indicated the role of P450 2E1 in the NADPH dependent lipid peroxidation in rat blood lymphocytes. The data indicating similarities in the NADPH dependent lipid peroxidation and NDMA-d activity in blood lymphocyte with the liver microsome have provided evidence that blood lymphocyte P450 2E1 could be used as a surrogate to monitor and predict hepatic levels of the enzyme. D 2002 Published by Elsevier Science Inc. Keywords: Lymphocyte; P450 2E1; Lipid peroxidation; Enzyme; CCl4; Inhibition
Introduction Cytochrome P450s (P450s) are the monooxygenases involved in the biotransformation of endogenous compounds like steroids and prostaglandins and exogenous compounds like drugs, carcinogens and *
Corresponding author. Fax: +91-522-228227. E-mail address: parmar_devendra1@ rediffmail.com (D. Parmar). 0024-3205/02/$ - see front matter D 2002 Published by Elsevier Science Inc. PII: S 0 0 2 4 - 3 2 0 5 ( 0 2 ) 0 2 0 8 4 - 2
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pesticides [1]. P450 2E1, an isoenzyme expressed in abundance in most humans, is specifically involved in the metabolism of procarcinogens like N-nitrosamines and toxic, low molecular weight solvents like benzene, styrene and a number of halocarbons [2]. P450 2E1, due to its existence predominantly in high spin form, also reduces dioxygen to reactive oxyradicals such as superoxide anion and hydrogen peroxide, which act as initiators of membrane lipid peroxidation [3–5]. In view of the ability of P450 2E1 to generate reactive oxygen intermediates and the known toxicity of these free oxygen radical species, P450 2E1 plays a key role in the pathogenesis of liver injury. CYP 2E1 activity can also be induced in animals by altered physiological states, including fasting and diabetes and by treatment with agents such as ethanol, acetone and isoniazid [3–5]. In recent years, there has been an interest to develop assays which can be used as biomarkers to detect and predict individuals at risk and partly explain the huge interindividual variations in responsiveness to drugs and chemicals. Lymphocytes have been used to develop noninvasive bioassays to screen human population for toxicant exposure and these cells have been used to determine exposure and susceptibility to the toxicants. Several of the P450 genes have been shown to be expressed in human blood lymphocytes [6–8]. Although P450s have been used as a biomarker of susceptibility with the individuals having variant genotypes found to be at greater risk to the toxicity of carcinogens, recently interest has been centered to develop and validate P450 mRNA expression as a biomarker to predict exposure of the environmental contaminants and their effects. Recent studies from our laboratory have shown similarities in the regulation of lymphocyte P450 1A1 with the liver isoenzyme and have suggested that P450 1A1 expression in peripheral blood lymphocytes can be used to monitor tissue levels of this enzyme [9]. Previous studies have reported the expression of P450 2E1 in lymphocytes of human and several animal species [10–12]. Using RT-PCR and immunoblot analysis, mRNA and protein expression of P450 2E1 has been reported in cultured lymphocytes of both humans and animals [7,10]. The expression of P450 2E1 was found to be induced in the lymphocytes of the individuals suffering from insulin dependent diabetes and in alcoholics [7,11]. Though the factors that affect the expression of P450 2E1 in liver could also affect their expression in lymphocytes, P450 2E1 mRNA may not always correspond to protein levels [13] and the low levels of mRNA in blood lymphocytes may cause amplification of erroneous gene products. No information is also available on the catalytic and functional activity of P450 2E1 in blood lymphocytes, hampering the successful utilisation of blood for monitoring tissue expression of the enzyme. Therefore, to understand if the blood lymphocyte P450 2E1 could serve as surrogates for monitoring hepatic changes of the enzyme, attempts were made to investigate the catalytic activity of P450 2E1 in lymphocytes by studying NADPH (basal) and CCl4 supported NADPH dependent lipid peroxidation and demethylation of N-nitrosodimethylamine (NDMA) in rat blood lymphocytes.
Material and methods Chemicals Histopaque 1077, 3-methylcholanthrene (MC), dimethyl formamide (DMF), dimethyl sulfoxide (DMSO), NADPH, N-nitrosodimethyl amine (NDMA), carbon tetrachloride (CCl4) and thiobarbituric acid (TBA) were obtained from Sigma, USA. Pyrazole and ethanol were obtained from Merck
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(India) Ltd. and Bengal Chemicals respectively. All other chemicals used were of highest purity commercially available and procured either from BDH (India) or SRL (India). Polyclonal antibody to rat hepatic P450 2E1 was gifted by Prof. Dr. Johannes Doehmer, Institute of Toxicology, Technical University, Munich, Germany. Animals and treatment Male albino Wistar rats (f 8 weeks old) were obtained from 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 Animal Care Committee of Industrial Toxicology Research Centre and Animal Experimentation was approved by the Ethical Committee of the Centre. The animals were divided into three groups of ten animals each. Rats in the first and second groups received a single dose of ethanol (0.8 ml/kg b.wt., i.p.) or pyrazole (200 mg/kg b.wt., i.p.) for four consecutive days. The rats in the third group were treated with acetone (5 ml/kg, given as 33% (v/v) acetone in 0.9% (w/v) NaCl orally) for two days. The animals in the fourth group served as the controls. Blood was drawn from the choroid plexus of the animals 16 hours after the last dose following which the animals were sacrificed and liver was immediately taken out and processed for isolation of microsomes as described earlier [14]. Isolation of lymphocytes Lymphocytes were isolated from the blood by a slight modification of the method of Boyum [15]. 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 g for 30 min at room temperature, the upper layer was discarded and the opaque interface containing the lymphocytes and negligible amount of monocytes was transferred into a clean centrifuge tube. After repeated washing of the lymphocytes with PBS and recentrifugation at 250 g, the resulting pellet was resuspended in 0.5 ml of PBS and used for enzyme estimation. The number of lymphocytes was counted using hemocytometer. Approximately 2–3 106 cells were present in 0.5 ml lymphocyte suspension. The viability of the cells was estimated by trypan blue exclusion test. Lipid peroxidation studies P450 2E1 dependent lipid peroxidation was studied by following the method of Masuda and Murano [16]. The optimum protein concentration and the optimum incubation time for lipid peroxidation in rat blood lymphocytes were determined by varying the protein concentration in the range of 50 Ag–1000 Ag and the incubation time for 10–30 mins. The 2.0 ml reaction mixture for NADPH-supported lipid peroxidation (endogenous lipid peroxidation) was initiated by the addition of NADPH and contained 1.0 mg liver microsomal or 700 Ag lymphocyte protein, 200 AM NADPH and 0.1 M potassium phosphate buffer, pH 7.5. The reaction mixture for CCl4 supported lipid peroxidation (exogenous lipid peroxidation) was initiated by CCl4 and contained 4.3 mM CCl4 in addition to the above. The reaction mixture containing CCl4 was kept in closed tubes and the reaction mixture with or without CCl4 was incubated at 37 jC for 20 min. The reaction was stopped by addition of 2.0 ml of 30% TCA and 0.2 ml of 5 M HCl. MDA was determined by thiobarbituric acid method of Ernster and Nordenbrand [17] using a molar extinction coefficient 1.56 105 M 1 cm 1 at 535 nm.
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Enzyme assay N-nitrosodimethylamine demethylase (NDMA-d) activity was assayed in blood lymphocytes and liver microsomes by a slight modification of the method of Castonguay et al. [18]. The assay mixture contained a suitable amount of lymphocytes or liver microsomes, 70.0 mM Tris-HCl, pH 7.4, 10 mM semicarbazide, 14 mM MgCl2, 215 mM KCl, 1 mM NADPH and 4 mM NDMA in 1.0 ml final volume. The reaction mixture was incubated at 37 jC 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 jC for 20 min and the HCHO formed was measured at 415 nm. In vitro studies with different solvents like DMF, DMSO and hexane, which are known to inhibit P450 2E1 dependent reactions in liver and polyclonal antibody raised against rat hepatic P450 2E1, were performed in blood lymphocytes and liver microsomes isolated from control or ethanol pretreated rats as described in our earlier studies [14,19]. Following preincubation with the above solvents for 20 min, NADPH was added to reaction mixture for measuring endogenous lipid peroxidation and NDMA-d activity or CCl4 was added to reaction mixture for determing exogenous lipid peroxidation and the reaction was allowed to continue for 20 min. MDA formed or enzyme activity was measured as described above.
Results Lipid peroxidation in rat blood lymphocytes Characterisation of the P450 dependent lipid peroxidation revealed that maximum NADPH dependent (basal) lipid peroxidation was observed when 700 Ag of lymphocyte protein was incubated for 20 minutes. Since subcellular fractionation or sonication of cells led to a decrease in MDA production, intact cells were used to study lipid peroxidation in lymphocytes (data not shown). Pretreatment with ethanol or pyrazole or acetone resulted in almost 2.0 fold and 2.5 fold increase in basal lipid peroxidation in lymphocytes and liver respectively (Table 1). In vitro addition of CCl4 to the control lymphocytes Table 1 NADPH dependent lipid peroxidation and NDMA-d activity in rat liver microsomes and blood lymphocytes Lipid peroxidationa Control Ethanol Pyrazole Acetone
NDMA-db
Lymphocytes
Liver
0.024 0.048 0.045 0.046
0.038 0.087 0.085 0.086
F F F F
0.001 0.002 * 0.003 * 0.003 *
All the values are mean F S.E. of 5 animals. a nmoles MDA/min/mg protein. b nmoles HCHO/min/mg protein. * p < 0.05 when compared to the controls.
F F F F
0.002 0.004 * 0.005 * 0.004 *
Lymphocytes
Liver
1.06 2.71 2.33 2.18
2.68 9.38 9.05 8.85
F F F F
0.003 0.006 * 0.001 * 0.002 *
F F F F
0.001 0.003 * 0.003 * 0.003 *
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produced almost 1.5–2.0 fold increase in NADPH dependent lipid peroxidation. Similar increase (2.0– 2.5 fold) in the NADPH dependent lipid peroxidation was observed on in vitro addition of CCl4 to the control liver microsomes (Table 2). In vitro addition of CCl4 to the lymphocytes isolated from ethanol or pyrazole or acetone pretreated rats produced almost 2.0 fold induction in NADPH dependent lipid peroxidation in lymphocytes whereas 4.0–5.0 fold induction was observed in induced liver microsomes on in vitro addition of CCl4 (Table 2). N-nitrosodimethylamine demethylase (NDMA-d) activity in rat blood lymphocytes Significant activity of NDMA-d, though less than that observed in the liver, was found in rat blood lymphocytes. Pretreatment of rats with ethanol or pyrazole or acetone resulted in significant induction of NDMA-d activity in rat blood lymphocytes. Approximately 2.0–2.5 fold increase in the NDMA-d activity was observed in blood lymphocytes after pretreatment with inducers as compared to 4–5 fold induction of NDMA-d activity in liver after exposure to these inducers (Table 1). Effect of inhibitors and anti-P450 2E1 on NADPH and CCl4 supported NADPH dependent lipid peroxidation Tables 3 and 4 summarise the in vitro effects of organic P450 2E1 inhibitors and anti P450 2E1 on the basal and CCl4 supported NADPH dependent lipid peroxidation in blood lymphocytes. Though the addition of DMF or hexane or DMSO to control lymphocytes produced inhibition of basal lipid peroxidation, significant inhibition was observed only with DMSO at the highest concentration, 10 5 M (Table 3). Likewise in lymphocytes isolated from ethanol pretreated rats, addition of DMF or DMSO or hexane produced a concentration dependent inhibition of lipid peroxidation, with significant inhibition occuring at the highest concentration (10 5 M) of the inhibitors. Similarly, in vitro addition of anti-P450 2E1 to the blood lymphocytes isolated from control rats produced significant inhibition of NADPH dependent lipid peroxidation at relatively higher concentrations while a significant concentration dependent inhibition was observed on in vitro addition to the blood lymphocytes isolated from ethanol pretreated animals. In contrast, addition of preimmune IgG to the lymphocytes isolated from control or induced animals did not produce any significant effect on the basal lipid peroxidation (Table 4). As observed with the blood lymphocytes, similar pattern of NADPH dependent lipid peroxidation was
Table 2 In vitro effect of CCl4 on NADPH dependent lipid peroxidation Lymphocytes NADPH Control Ethanol Pyrazole Acetone
0.025 0.046 0.043 0.048
F F F F
0.001 0.002 0.003 0.003
Liver NADPH + CCl4
NADPH
0.041 0.080 0.084 0.085
0.035 0.085 0.080 0.082
Values are expressed in nmoles MDA/min/mg protein. Values represent data F S.E. of 3 experiments. * p < 0.05 when compared to the controls.
F F F F
0.004 * 0.005 * 0.005 * 0.005 *
F F F F
NADPH + CCl4 0.002 0.004 * 0.003 * 0.003 *
0.08 0.41 0.38 0.40
F F F F
0.001 * 0.004 * 0.004 * 0.004 *
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Table 3 Effect of P450 2E1 inhibitors on NADPH and CCl4 supported NADPH dependent lipid peroxidation in whole blood lymphocytes and liver microsomes Lymphocyte
Liver
None EtOH
Ethanol
Control
Ethanol
NADPH
CCl4
NADPH
CCl4
NADPH
CCl4
NADPH
CCl4
0.028 F 0.002 0.026 F 0.003
0.039 F 0.003 0.037 F 0.002
0.058 F 0.001 0.055 F 0.002
0.098 F 0.002 0.093 F 0.002
0.038 F 0.002 0.036 F 0.002
0.080 F 0.001 0.077 F 0.001
0.087 F 0.004 0.085 F 0.004
0.43 F 0.04 0.41 F 0.004
DMSO 10 7 M 0.025 F 0.004 0.035 F 0.001 0.044 F 0.004 0.039 F 0.002 * 0.034 F 0.002 0.073 F 0.002 0.073 F 0.004 0.27 F 0.003 * 10 6 M 0.021 F 0.004 0.029 F 0.002 0.041 F 0.003 0.024 F 0.001 * 0.031 F 0.002 0.055 F 0.002 0.068 F 0.004 0.22 F 0.003 * 10 5 M 0.016 F 0.001 * 0.017 F 0.005 * 0.027 F 0.002 * 0.018 F 0.002 * 0.021 F 0.001 * 0.031 F 0.002 * 0.044 F 0.004 * 0.13 F 0.003 * DMF 10 7 M 0.024 F 0.001 10 6 M 0.020 F 0.002 10 5 M 0.018 F 0.001
0.032 F 0.003 0.050 F 0.002 0.046 F 0.004 * 0.030 F 0.002 0.074 F 0.002 0.078 F 0.004 0.25 F 0.003 * 0.021 F 0.002 * 0.038 F 0.003 0.024 F 0.006 * 0.030 F 0.002 0.057 F 0.003 0.059 F 0.003 * 0.20 F 0.003 * 0.018 F 0.002 * 0.021 F 0.002 * 0.018 F 0.002 * 0.020 F 0.001 * 0.035 F 0.003 * 0.039 F 0.003 * 0.14 F 0.004 *
Hexane 10 7 M 0.023 F 0.001 10 6 M 0.020 F 0.001 10 5 M 0.017 F 0.002
0.031 F 0.002 0.048 F 0.001 0.044 F 0.002 * 0.034 F 0.002 0.074 F 0.003 0.075 F 0.004 0.25 F 0.003 * 0.018 F 0.002 * 0.027 F 0.002 0.024 F 0.003 * 0.026 F 0.001 0.051 F 0.003 0.071 F 0.004 0.20 F 0.003 * 0.015 F 0.003 * 0.020 F 0.001 * 0.014 F 0.002 * 0.020 F 0.001 * 0.039 F 0.003 * 0.047 F 0.003 * 0.16 F 0.003 *
Values are expressed in nmoles MDA/min/mg protein and represent data F S.E. of 3 experiments. * p < 0.05 when compared to the controls.
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Control
Lymphocyte
Liver
Control
Ethanol
Control
Ethanol
NADPH
CCl4
NADPH
CCl4
NADPH
CCl4
NADPH
CCl4
0.028 F 0.002
0.039 F 0.003
0.058 F 0.001
0.098 F 0.002
0.038 F 0.002
0.080 F 0.001
0.087 F 0.04
0.43 F 0.04
Preimmune IgG 0.3 mg 0.026 F 0.002 0.5 mg 0.025 F 0.002 0.8 mg 0.024 F 0.002
0.036 F 0.001 0.035 F 0.001 0.033 F 0.004
0.055 F 0.003 0.053 F 0.003 0.050 F 0.002
0.090 F 0.001 0.088 F 0.001 0.087 F 0.001
0.036 F 0.002 0.034 F 0.003 0.033 F 0.002
0.076 F 0.002 0.075 F 0.003 0.073 F 0.003
0.081 F 0.003 0.080 F 0.004 0.075 F 0.003
0.39 F 0.03 0.38 F 0.02 0.37 F 0.02
None
Anti-P4502E1 0.3 mg 0.019 F 0.003 0.018 F 0.002 * 0.029 F 0.001 0.033 F 0.001 * 0.024 F 0.003 0.053 F 0.002 0.054 F 0.002 0.23 F 0.02 * 0.5 mg 0.013 F 0.001 * 0.012 F 0.001 * 0.024 F 0.001 0.025 F 0.001 * 0.021 F 0.003 0.038 F 0.002 * 0.050 F 0.002 0.18 F 0.02 * 0.8 mg 0.008 F 0.001 * 0.008 F 0.002 * 0.017 F 0.003 * 0.008 F 0.001 * 0.018 F 0.002 * 0.031 F 0.002 * 0.039 F 0.002 * 0.16 F 0.02 * Values are expressed in nmoles MDA/min/mg protein and represent data F S.E. of 3 experiments. * p < 0.05 when compared to the controls.
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Table 4 Effect of P450 2E1 inhibitors on NADPH and CCl4 supported NADPH dependent lipid peroxidation in whole rat blood lymphocytes and liver microsomes
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observed on in vitro addition of these inhibitors to liver microsomes isolated from control or ethanol pretreated rats (Tables 3 and 4). Similar to that observed with basal lipid peroxidation, DMF, hexane or DMSO produced a significant inhibition of CCl4 supported NADPH dependent lipid peroxidation in the control lymphocytes at relatively higher concentrations of the inhibitors. As observed with basal lipid peroxidation, addition of inhibitors produced a concentration dependent inhibition of CCl4 supported NADPH dependent lipid peroxidation in lymphocytes isolated from ethanol pretreated rats with almost 80–90% inhibition occurring at the higher concentrations. All the solvents were found to be equally effective in inhibiting CCl4 supported NADPH dependent lipid peroxidation (Table 3). Likewise, addition of anti-P450 2E1 produced inhibition of CCl4 supported NADPH dependent lipid peroxidation when added to control or ethanol induced lymphocytes, though the inhibition was much more significant at all the concentrations in blood lymphocytes isolated from ethanol pretreated animals. In vitro addition of preimmune- IgG to the lymphocytes isolated from control or ethanol pretreated rats did not produce any significant change in the basal or CCl4 supported NADPH dependent lipid peroxidation (Table 4). Almost similar pattern of inhibition of CCl4 supported NADPH dependent lipid peroxidation was observed when the inhibitors or Table 5 Effect of P450 2E1 inhibitors on NDMA-d activity in rat liver and blood lymphocytes Lymphocytes
Liver
Control
Ethanol
Control
Ethanol
Sample MeOH
0.98 F 0.04 0.92 F 0.02
2.35 F 0.01 2.29 F 0.06
2.68 F 0.01 2.56 F 0.04
9.38 F 0.03 8.64 F 0.01
DMF 10 3 M 10 2 M 10 1 M
0.85 F 0.02 0.74 F 0.03 0.29 F 0.03 *
1.81 F 0.02 0.96 F 0.02 * 0.54 F 0.01 *
2.04 F 0.02 1.96 F 0.01 1.2 F 0.02 *
6.04 F 0.03 3.23 F 0.02 * 1.9 F 0.02
DMSO 10 3 M 10 2 M 10 1 M
0.78 F 0.02 0.60 F 0.02 0.25 F 0.01 *
0.97 F 0.03 0.68 F 0.02 * 0.51 F 0.02 *
1.78 F 0.01 1.48 F 0.03 0.81 F 0.02 *
3.44 F 0.04 * 2.3 F 0.06 * 1.16 F 0.02 *
Hexane 10 3 M 10 2 M 10 1 M
0.83 F 0.03 0.66 F 0.02 0.37 F 0.02 *
1.16 F 0.04 0.82 F 0.02 * 0.42 F 0.01 *
2.18 F 0.02 2.02 F 0.02 1.28 F 0.03 *
6.2 F 0.01 3.36 F 0.03 * 2.07 F 0.02 *
Preimmune IgG 0.3 mg 0.8 mg
0.80 F 0.01 0.79 F 0.01
2.12 F 0.03 2.06 F 0.02
2.54 F 0.02 2.48 F 0.02
8.42 F 0.01 8.39 F 0.03
Anti P4502E1 0.3 mg 0.8 mg
0.51 F 0.01 0.28 F 0.02 *
0.50 F 0.03 * 0.33 F 0.02 *
1.52 F 0.01 0.68 F 0.01 *
1.76 F 0.02 * 0.42 F 0.01 *
Values are expressed in nmoles HCHO/min/mg protein. * p < 0.05 when compared with control.
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anti-P450 2E1 was added in vitro to the reaction mixture containing liver microsomes isolated from control or ethanol pretreated rats (Tables 3 and 4). Effect of organic inhibitors and anti-P450 2E1 on NDMA-d activity in blood lymphocytes The effect of solvents for eg. DMF, DMSO and hexane and anti-P450 2E1 on the activity of NDMAd, when added in vitro to the lymphocytes isolated from control or ethanol pretreated rats is summarised in Table 5. Addition of DMSO or DMF or hexane or anti-P450 2E1 to the blood lymphocytes isolated from control rats produced a concentration dependent inhibition of the enzyme activity with significant inhibition occuring only at higher concentrations. Similar to that seen in the lymphocytes, significant inhibition of the enzyme activity was observed in liver microsomes with higher concentrations of the inhibitors. Addition of preimmune IgG had no significant effect when added in vitro to liver microsomes or blood lymphocytes isolated from control rats (Table 5). On the other hand, in vitro addition of DMSO or DMF or hexane or anti-P450 2E1 to the blood lymphocytes isolated from ethanol pretreated rats produced a significant concentration dependent inhibition of the enzyme activity with almost 80% inhibition occuring with all the solvents or anti-P450 2E1 at the highest concentrations. Similar pattern of inhibition of the enzyme activity was observed when these inhibitors were added to the liver microsomes isolated from ethanol pretreated rats (Table 5).
Discussion Raucy et al [19] have shown that though considerable variations exist amongst the laboratories in the detection of P450s in blood lymphocytes, it has been shown that P450 2E1 and P450 2D6 exhibit the highest expression and greatest inter-laboratory reproducibility and the concentrations of these P450s in blood lymphocytes may reflect the in vivo activity of the corresponding liver isoenzymes. Even though the physiological role of P450s in blood is highly speculative at present, NADPH dependent (basal) lipid peroxidation and demethylation of N-nitrosodimethylamine in blood lymphocytes have indicated that P450 2E1 expressed in blood lymphocytes are functionally and catalytically active. As observed in the liver, several fold induction in the activity of NDMA-d by pretreatment with ethanol or acetone or pyrazole, have indicated selective enrichment of P450 2E1 isoenzymes. Since higher rates of NADH oxidase activity and NADPH- supported lipid peroxidation occur in liver microsomes of animals treated with P450 2E1 inducers, the enrichment of P450 2E1 in blood lymphocytes following pretreatment with ethanol or pyrazole or acetone may lead to enhanced rate of generation of active oxygen species resulting in increase in the rate of basal lipid peroxidation in peripheral blood lymphocytes [20]. Significant increase in the basal lipid peroxidation in rat blood lymphocytes on in vitro addition of CCl4 have indicated similarities in the NADPH dependent lipid peroxidation in rat blood lymphocytes with the liver. In the liver, carbon tetrachloride has been shown to be homolytically cleaved by P450 2E1 into trichloromethyl radicals and chloride anion which initiate a free radical attack on the membrane lipids leading to peroxidative decomposition of structural lipids [21]. Pretreatment of ethanol, which leads to the selective enrichment of P450 2E1, increases the formation of these radicals from CCl4 and potentiates CCl4 induced liver damage [22]. Similar to that seen in liver microsomes, an increase in NADPH dependent lipid peroxidation was observed on in vitro addition of CCl4 to lymphocytes isolated from control or ethanol pretreated rats. Though the involvement of P450 isoforms other than P450 2E1,
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such as P450 1A2 and P450 2B1 etc., in the CCl4 supported NADPH dependent lipid peroxidation cannot be ruled out in control blood lymphocytes, P450 2E1, which is selectively induced following treatment with ethanol in blood lymphocytes, maybe solely involved to a large extent, in the stimulation of CCl4 supported NADPH lipid peroxidation in blood lymphocytes isolated from ethanol pretreated rats. Similar mechanisms have been shown to occur in liver microsomes isolated from control and ethanol pretreated rats [23]. In vitro studies have also indicated similarities in the catalytic activity of P450 2E1 in blood lymphocytes with the liver isoenzymes. Significant inhibition of NDMA-d activity and NADPHdependent lipid peroxidation on in vitro addition of P450 2E1 inhibitors such as DMSO, DMF or hexane or anti-P450 2E1 to the blood lymphocytes isolated from control rats have indicated that as in liver, P450 2E1 is expressed in control blood lymphocytes and catalyses the activity of NDMA-d and NADPH dependent lipid peroxidation. Similarly, as observed in liver microsomes, greater magnitude of inhibition in the activity of NDMA-d and NADPH dependent lipid peroxidation on addition of organic inhibitors or anti-P450 2E1 to blood lymphocytes isolated from ethanol pretreated animals have indicated enrichment of P450 2E1 in blood lymphocytes following exposure to ethanol. Further evidence indicating similarities in the expression and catalytic activity of P450 2E1 in blood lymphocytes with the liver isoenzyme was provided by significant increase in the magnitude of inhibition of CCl4 supported NADPH dependent lipid peroxidation on in vitro addition of either of DMF, DMSO or hexane or anti-P450 2E1 to lymphocytes isolated from control or ethanol pretreated rats. Even though enzymes other than P450s such as catalase maybe involved in metabolising DMF or DMSO or hexane [24–26] or P450s other P450 2E1 in metabolising CCl4, almost 80–90% inhibition occurring in ethanol induced lymphocytes have further shown that like in liver, CCl4 is cleaved to free radicals by P450 2E1 expressed in blood lymphocytes leading to the initiation of lipid peroxidation. In conclusion, the results of the present study have shown that P450 2E1 expressed in blood lymphocytes catalyses the basal and CCl4 supported NADPH dependent lipid peroxidation and activity of NDMA-d in rat blood lymphocytes. The similarities in the factors regulating and affecting the catalytic activity of P450 2E1 in blood lymphocyte with the liver isoenzyme have further demonstrated that P450 2E1 in blood lymphocytes could be used to monitor and predict the hepatic levels of the enzyme. Furthermore, characterisation of P450 2E1 dependent lipid peroxidation in blood lymphocytes could be of immense significance in identifying intracellular oxidative damage in the alcoholic subjects and the individuals who could be at greater risk to ethanol induced hepatotoxicity. Acknowledgements AD is grateful to CSIR, New Delhi for providing Senior Research Fellowship. The technical assistance of Mr. Kanahiya Lal and Mr. B.S. Pandey and computer assistance of Mr. Mohd. Aslam are gratefully acknowledged. ITRC Communication No. 2201. References [1] Gonzalez FJ. Molecular genetics of the P450 superfamily. Pharmacol Ther 1990;45:1 – 38. [2] Koop DR. Oxidative and reductive metabolism by cytochrome P450 2E1. FASEB J 1992;6:724 – 30.
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[3] Carriere V, Berthou F, Baird S, Belloc C, Beaune P, de Waziers I. Human cytochrome P450 2E1 (CYP 2E1): from genotype to phenotype. Pharmacogenetics 1996;6:203 – 11. [4] Ma Q, Dannan GA, Guengerich FP, Yang CS. Similarities and differences in the regulation of hepatic cytochrome P450 enzymes by diabetes and fasting in male rats. Biochem Pharmacol 1989;38:3179 – 84. [5] Koop DR, Crump BL, Nordbloom GO, Coon MJ. Immunochemical evidence for induction of the alcohol oxidising cytochrome - P450 of rat liver microsomes by diverse agents: ethanol, imidazole, trichloroethylene, acetone, pyrazole and isoniazid. Proc Natl Acad Sci USA 1985;82:4065 – 9. [6] Petushkova NA, Bachmanova GI, Archakov AI. 7-Alkoxyphenoxazone and 7-Alkoxycoumarin O-Dealkylation by human peripheral blood lymphocytes. In vitro Toxicology 1996;9:291 – 5. [7] Raucy JL, Schultz E, Wester M, Arora S, Johnston D, Ondahl J, Carpenter S. Human lymphocyte cytochrome P450 2E1, a putative marker for alcohol mediated changes in hepatic chlorzooxazone activity. Drug Metab Dispos 1997;25:1429 – 35. [8] Baron JM, Zwaldo-Klarwasser G, Jugert F, Hamann W, Rubben A, Mukhtar H, Merk HF. Cytochrome P450 1B1: A major P450 isoenzyme in human blood monocytes and macrophase subsets. Biochem Pharmacol 1998;56:1105 – 10. [9] Dey A, Parmar D, Dayal M, Dhawan A, Seth PK. Cytochrome P450 1A1 (CYP 1A1) in blood lymphocytes: Evidence for catalytic activity and mRNA expression. Life Sci 2001;69:383 – 93. [10] Scobbie AE, Mason HJ. Measurement of immunoreactive cytochrome P450 2E1 in human leukocytes. Biomarkers 1999;4:311 – 7. [11] Song BJ, Veech RL, Sanger P. Cytochrome P450 2E1 is elevated in lymphocytes from poorly controlled insulin dependent diabetes. J Clin Endocrinol Metab 1990;71:1036 – 40. [12] Raucy JL, Curley G, Carpenter SP. Use of lymphocytes for assessing ethanol mediated alterations in the expression of hepatic cytochrome P450 2E1. Alcoholism: Clinical and Experimental Research 1995;19:1369 – 75. [13] Roberts BJ, Song BJ, Soh Y, Park SS, Shoaf SE. Ethanol induces CYP 2E1 by protein stabilization- Role of ubiquitin conjugation in the rapid degradation of CYP 2E1. J Biol Chem 1995;270:29632 – 5. [14] Parmar D, Dhawan A, Dayal M, Seth PK. Immuochemical and biochemical evidence for expression of phenobarbital- and 3-methylcholanthrene inducible isoenzymes of cytochrome P450 in rat brain. Int J Toxicol 1998;17:619 – 30. [15] Boyum A. Separation of leukocytes from blood and bone marrow. Scand J Clin Invest 1963;21:77 – 90. [16] Masuda Y, Murano T. Carbon tetrachloride - induced lipid peroxidation of rat liver microsomes in vitro. Biochem Pharmacol 1977;26:2275 – 82. [17] Ernster L, Nordenbrand K. The Thiobarbituric Acid Assay. In: Estabrook RW, Pullman ME, editors. Methods in Enzymology, vol. 10. New York: Academic Press; 1967. p. 574. [18] Castonguay A, Pepin P, Briere N. Modulation of 4-(methylnitrosamino)-1-(3-pyridyl)-1 butanone demethylation and denitrosation by rat liver microsomes. Cancer Lett 1991;59:67 – 74. [19] Dhawan A, Parmar D, Dayal M, Seth PK. Cytochrome P450 (P450) isoenzyme specific dealkylation of alkoxyresorufins in rat brain microsomes. Mol Cell Biochem 1999;200:169 – 76. [20] Raucy JL, Ingelman Sundberg M. Drug metabolising enzymes in lymphocytes. J Biochem Mol Toxicol 1999;13:223 – 6. [21] Lieber CS. Cytochrome P450 2E1: Its physiological and pathological role. Physiol Rev 1997;77:517 – 44. [22] Recknagel RO, Glende Jr EA, Hruskewycz AM. Chemical Mechanisms in CCl4 toxicity. In: Pryer WE, editor. Free Radicals in Biology, vol. III. London: Academic Press Inc.; 1977. p. 97 – 132. [23] Lindros KO, Cai Y, Pentilla KE. Role of ethanol- inducible cytochrome P450 IIE1 in carbon tetrachloride induced damage to centrilobular hepatocytes from ethanol treated rats. Hepatology 1990;12:1092 – 7. [24] Johansson I, Ekstrom G, Scholte B, Puzycki D, Jornvall H, Ingelman-Sundberg M. Ethanol, fasting and acetone inducible cytochromes P450 in rat liver: Regulation and characteristics of enzymes belonging to the IIB and IIE gene subfamilies. Biochemistry 1988;27:1925 – 34. [25] Scailteur V, Lauwerys R. In vivo and in vitro oxidative biotransformation of dimethylformamide in rat. Chem Biol Interact 1984;50:327 – 37. [26] Rosenblum WI, Wei EP, Kontos AH. Dimethylsulfoxide and ethanol, commonly used diluents, present dilation of pial arterioles by openers of KATP ion channels. Eur J Pharmacol 2001;430:101 – 6.
Cytochrome P450 2E1 dependent catalytic activity and lipid peroxidation in rat blood lymphocytes A. Dey a, D. Parmar a,*, A. Dhawan a, D. Dash b, P.K. Seth a a
Developmental Toxicology Division, Industrial Toxicology Research Centre, P.O. Box 80, M.G. Marg, Lucknow 226 001, India b Department of Biochemistry, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221 005, India Received 23 April 2001; received in revised form 12 October 2001; accepted 31 May 2002
Abstract To investigate the similarities in the catalytic activity of blood lymphocyte P450 2E1 in blood lymphocyte with the liver isoenzyme, NADPH dependent lipid peroxidation and activity of N-nitrosodimethyamine demethylase (NDMA-d) was studied in rat blood lymphocytes. Blood lymphocytes were found to catalyse NADPH dependent (basal) lipid peroxidation and demethylation of N-nitrosodimethylamine (NDMA). Pretreatment with ethanol or pyrazole or acetone resulted in significant increase in the NADPH dependent lipid peroxidation and the activity of NDMA-d in blood lymphocytes and liver microsomes. In vitro addition of CCl4 to the blood lymphocytes isolated from control or ethanol pretreated rats resulted in an increase in the NADPH dependent lipid peroxidation. Significant inhibition of the basal and CCl4 supported NADPH dependent lipid peroxidation and NDMA-d activity in blood lymphocytes isolated from control or ethanol pretreated rats by dimethyl formamide or dimethyl sulfoxide or hexane, solvents known to inhibit P450 2E1 catalysed reactions in liver and anti- P450 2E1, have indicated the role of P450 2E1 in the NADPH dependent lipid peroxidation in rat blood lymphocytes. The data indicating similarities in the NADPH dependent lipid peroxidation and NDMA-d activity in blood lymphocyte with the liver microsome have provided evidence that blood lymphocyte P450 2E1 could be used as a surrogate to monitor and predict hepatic levels of the enzyme. D 2002 Published by Elsevier Science Inc. Keywords: Lymphocyte; P450 2E1; Lipid peroxidation; Enzyme; CCl4; Inhibition
Introduction Cytochrome P450s (P450s) are the monooxygenases involved in the biotransformation of endogenous compounds like steroids and prostaglandins and exogenous compounds like drugs, carcinogens and *
Corresponding author. Fax: +91-522-228227. E-mail address: parmar_devendra1@ rediffmail.com (D. Parmar). 0024-3205/02/$ - see front matter D 2002 Published by Elsevier Science Inc. PII: S 0 0 2 4 - 3 2 0 5 ( 0 2 ) 0 2 0 8 4 - 2
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pesticides [1]. P450 2E1, an isoenzyme expressed in abundance in most humans, is specifically involved in the metabolism of procarcinogens like N-nitrosamines and toxic, low molecular weight solvents like benzene, styrene and a number of halocarbons [2]. P450 2E1, due to its existence predominantly in high spin form, also reduces dioxygen to reactive oxyradicals such as superoxide anion and hydrogen peroxide, which act as initiators of membrane lipid peroxidation [3–5]. In view of the ability of P450 2E1 to generate reactive oxygen intermediates and the known toxicity of these free oxygen radical species, P450 2E1 plays a key role in the pathogenesis of liver injury. CYP 2E1 activity can also be induced in animals by altered physiological states, including fasting and diabetes and by treatment with agents such as ethanol, acetone and isoniazid [3–5]. In recent years, there has been an interest to develop assays which can be used as biomarkers to detect and predict individuals at risk and partly explain the huge interindividual variations in responsiveness to drugs and chemicals. Lymphocytes have been used to develop noninvasive bioassays to screen human population for toxicant exposure and these cells have been used to determine exposure and susceptibility to the toxicants. Several of the P450 genes have been shown to be expressed in human blood lymphocytes [6–8]. Although P450s have been used as a biomarker of susceptibility with the individuals having variant genotypes found to be at greater risk to the toxicity of carcinogens, recently interest has been centered to develop and validate P450 mRNA expression as a biomarker to predict exposure of the environmental contaminants and their effects. Recent studies from our laboratory have shown similarities in the regulation of lymphocyte P450 1A1 with the liver isoenzyme and have suggested that P450 1A1 expression in peripheral blood lymphocytes can be used to monitor tissue levels of this enzyme [9]. Previous studies have reported the expression of P450 2E1 in lymphocytes of human and several animal species [10–12]. Using RT-PCR and immunoblot analysis, mRNA and protein expression of P450 2E1 has been reported in cultured lymphocytes of both humans and animals [7,10]. The expression of P450 2E1 was found to be induced in the lymphocytes of the individuals suffering from insulin dependent diabetes and in alcoholics [7,11]. Though the factors that affect the expression of P450 2E1 in liver could also affect their expression in lymphocytes, P450 2E1 mRNA may not always correspond to protein levels [13] and the low levels of mRNA in blood lymphocytes may cause amplification of erroneous gene products. No information is also available on the catalytic and functional activity of P450 2E1 in blood lymphocytes, hampering the successful utilisation of blood for monitoring tissue expression of the enzyme. Therefore, to understand if the blood lymphocyte P450 2E1 could serve as surrogates for monitoring hepatic changes of the enzyme, attempts were made to investigate the catalytic activity of P450 2E1 in lymphocytes by studying NADPH (basal) and CCl4 supported NADPH dependent lipid peroxidation and demethylation of N-nitrosodimethylamine (NDMA) in rat blood lymphocytes.
Material and methods Chemicals Histopaque 1077, 3-methylcholanthrene (MC), dimethyl formamide (DMF), dimethyl sulfoxide (DMSO), NADPH, N-nitrosodimethyl amine (NDMA), carbon tetrachloride (CCl4) and thiobarbituric acid (TBA) were obtained from Sigma, USA. Pyrazole and ethanol were obtained from Merck
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(India) Ltd. and Bengal Chemicals respectively. All other chemicals used were of highest purity commercially available and procured either from BDH (India) or SRL (India). Polyclonal antibody to rat hepatic P450 2E1 was gifted by Prof. Dr. Johannes Doehmer, Institute of Toxicology, Technical University, Munich, Germany. Animals and treatment Male albino Wistar rats (f 8 weeks old) were obtained from 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 Animal Care Committee of Industrial Toxicology Research Centre and Animal Experimentation was approved by the Ethical Committee of the Centre. The animals were divided into three groups of ten animals each. Rats in the first and second groups received a single dose of ethanol (0.8 ml/kg b.wt., i.p.) or pyrazole (200 mg/kg b.wt., i.p.) for four consecutive days. The rats in the third group were treated with acetone (5 ml/kg, given as 33% (v/v) acetone in 0.9% (w/v) NaCl orally) for two days. The animals in the fourth group served as the controls. Blood was drawn from the choroid plexus of the animals 16 hours after the last dose following which the animals were sacrificed and liver was immediately taken out and processed for isolation of microsomes as described earlier [14]. Isolation of lymphocytes Lymphocytes were isolated from the blood by a slight modification of the method of Boyum [15]. 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 g for 30 min at room temperature, the upper layer was discarded and the opaque interface containing the lymphocytes and negligible amount of monocytes was transferred into a clean centrifuge tube. After repeated washing of the lymphocytes with PBS and recentrifugation at 250 g, the resulting pellet was resuspended in 0.5 ml of PBS and used for enzyme estimation. The number of lymphocytes was counted using hemocytometer. Approximately 2–3 106 cells were present in 0.5 ml lymphocyte suspension. The viability of the cells was estimated by trypan blue exclusion test. Lipid peroxidation studies P450 2E1 dependent lipid peroxidation was studied by following the method of Masuda and Murano [16]. The optimum protein concentration and the optimum incubation time for lipid peroxidation in rat blood lymphocytes were determined by varying the protein concentration in the range of 50 Ag–1000 Ag and the incubation time for 10–30 mins. The 2.0 ml reaction mixture for NADPH-supported lipid peroxidation (endogenous lipid peroxidation) was initiated by the addition of NADPH and contained 1.0 mg liver microsomal or 700 Ag lymphocyte protein, 200 AM NADPH and 0.1 M potassium phosphate buffer, pH 7.5. The reaction mixture for CCl4 supported lipid peroxidation (exogenous lipid peroxidation) was initiated by CCl4 and contained 4.3 mM CCl4 in addition to the above. The reaction mixture containing CCl4 was kept in closed tubes and the reaction mixture with or without CCl4 was incubated at 37 jC for 20 min. The reaction was stopped by addition of 2.0 ml of 30% TCA and 0.2 ml of 5 M HCl. MDA was determined by thiobarbituric acid method of Ernster and Nordenbrand [17] using a molar extinction coefficient 1.56 105 M 1 cm 1 at 535 nm.
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Enzyme assay N-nitrosodimethylamine demethylase (NDMA-d) activity was assayed in blood lymphocytes and liver microsomes by a slight modification of the method of Castonguay et al. [18]. The assay mixture contained a suitable amount of lymphocytes or liver microsomes, 70.0 mM Tris-HCl, pH 7.4, 10 mM semicarbazide, 14 mM MgCl2, 215 mM KCl, 1 mM NADPH and 4 mM NDMA in 1.0 ml final volume. The reaction mixture was incubated at 37 jC 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 jC for 20 min and the HCHO formed was measured at 415 nm. In vitro studies with different solvents like DMF, DMSO and hexane, which are known to inhibit P450 2E1 dependent reactions in liver and polyclonal antibody raised against rat hepatic P450 2E1, were performed in blood lymphocytes and liver microsomes isolated from control or ethanol pretreated rats as described in our earlier studies [14,19]. Following preincubation with the above solvents for 20 min, NADPH was added to reaction mixture for measuring endogenous lipid peroxidation and NDMA-d activity or CCl4 was added to reaction mixture for determing exogenous lipid peroxidation and the reaction was allowed to continue for 20 min. MDA formed or enzyme activity was measured as described above.
Results Lipid peroxidation in rat blood lymphocytes Characterisation of the P450 dependent lipid peroxidation revealed that maximum NADPH dependent (basal) lipid peroxidation was observed when 700 Ag of lymphocyte protein was incubated for 20 minutes. Since subcellular fractionation or sonication of cells led to a decrease in MDA production, intact cells were used to study lipid peroxidation in lymphocytes (data not shown). Pretreatment with ethanol or pyrazole or acetone resulted in almost 2.0 fold and 2.5 fold increase in basal lipid peroxidation in lymphocytes and liver respectively (Table 1). In vitro addition of CCl4 to the control lymphocytes Table 1 NADPH dependent lipid peroxidation and NDMA-d activity in rat liver microsomes and blood lymphocytes Lipid peroxidationa Control Ethanol Pyrazole Acetone
NDMA-db
Lymphocytes
Liver
0.024 0.048 0.045 0.046
0.038 0.087 0.085 0.086
F F F F
0.001 0.002 * 0.003 * 0.003 *
All the values are mean F S.E. of 5 animals. a nmoles MDA/min/mg protein. b nmoles HCHO/min/mg protein. * p < 0.05 when compared to the controls.
F F F F
0.002 0.004 * 0.005 * 0.004 *
Lymphocytes
Liver
1.06 2.71 2.33 2.18
2.68 9.38 9.05 8.85
F F F F
0.003 0.006 * 0.001 * 0.002 *
F F F F
0.001 0.003 * 0.003 * 0.003 *
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produced almost 1.5–2.0 fold increase in NADPH dependent lipid peroxidation. Similar increase (2.0– 2.5 fold) in the NADPH dependent lipid peroxidation was observed on in vitro addition of CCl4 to the control liver microsomes (Table 2). In vitro addition of CCl4 to the lymphocytes isolated from ethanol or pyrazole or acetone pretreated rats produced almost 2.0 fold induction in NADPH dependent lipid peroxidation in lymphocytes whereas 4.0–5.0 fold induction was observed in induced liver microsomes on in vitro addition of CCl4 (Table 2). N-nitrosodimethylamine demethylase (NDMA-d) activity in rat blood lymphocytes Significant activity of NDMA-d, though less than that observed in the liver, was found in rat blood lymphocytes. Pretreatment of rats with ethanol or pyrazole or acetone resulted in significant induction of NDMA-d activity in rat blood lymphocytes. Approximately 2.0–2.5 fold increase in the NDMA-d activity was observed in blood lymphocytes after pretreatment with inducers as compared to 4–5 fold induction of NDMA-d activity in liver after exposure to these inducers (Table 1). Effect of inhibitors and anti-P450 2E1 on NADPH and CCl4 supported NADPH dependent lipid peroxidation Tables 3 and 4 summarise the in vitro effects of organic P450 2E1 inhibitors and anti P450 2E1 on the basal and CCl4 supported NADPH dependent lipid peroxidation in blood lymphocytes. Though the addition of DMF or hexane or DMSO to control lymphocytes produced inhibition of basal lipid peroxidation, significant inhibition was observed only with DMSO at the highest concentration, 10 5 M (Table 3). Likewise in lymphocytes isolated from ethanol pretreated rats, addition of DMF or DMSO or hexane produced a concentration dependent inhibition of lipid peroxidation, with significant inhibition occuring at the highest concentration (10 5 M) of the inhibitors. Similarly, in vitro addition of anti-P450 2E1 to the blood lymphocytes isolated from control rats produced significant inhibition of NADPH dependent lipid peroxidation at relatively higher concentrations while a significant concentration dependent inhibition was observed on in vitro addition to the blood lymphocytes isolated from ethanol pretreated animals. In contrast, addition of preimmune IgG to the lymphocytes isolated from control or induced animals did not produce any significant effect on the basal lipid peroxidation (Table 4). As observed with the blood lymphocytes, similar pattern of NADPH dependent lipid peroxidation was
Table 2 In vitro effect of CCl4 on NADPH dependent lipid peroxidation Lymphocytes NADPH Control Ethanol Pyrazole Acetone
0.025 0.046 0.043 0.048
F F F F
0.001 0.002 0.003 0.003
Liver NADPH + CCl4
NADPH
0.041 0.080 0.084 0.085
0.035 0.085 0.080 0.082
Values are expressed in nmoles MDA/min/mg protein. Values represent data F S.E. of 3 experiments. * p < 0.05 when compared to the controls.
F F F F
0.004 * 0.005 * 0.005 * 0.005 *
F F F F
NADPH + CCl4 0.002 0.004 * 0.003 * 0.003 *
0.08 0.41 0.38 0.40
F F F F
0.001 * 0.004 * 0.004 * 0.004 *
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Table 3 Effect of P450 2E1 inhibitors on NADPH and CCl4 supported NADPH dependent lipid peroxidation in whole blood lymphocytes and liver microsomes Lymphocyte
Liver
None EtOH
Ethanol
Control
Ethanol
NADPH
CCl4
NADPH
CCl4
NADPH
CCl4
NADPH
CCl4
0.028 F 0.002 0.026 F 0.003
0.039 F 0.003 0.037 F 0.002
0.058 F 0.001 0.055 F 0.002
0.098 F 0.002 0.093 F 0.002
0.038 F 0.002 0.036 F 0.002
0.080 F 0.001 0.077 F 0.001
0.087 F 0.004 0.085 F 0.004
0.43 F 0.04 0.41 F 0.004
DMSO 10 7 M 0.025 F 0.004 0.035 F 0.001 0.044 F 0.004 0.039 F 0.002 * 0.034 F 0.002 0.073 F 0.002 0.073 F 0.004 0.27 F 0.003 * 10 6 M 0.021 F 0.004 0.029 F 0.002 0.041 F 0.003 0.024 F 0.001 * 0.031 F 0.002 0.055 F 0.002 0.068 F 0.004 0.22 F 0.003 * 10 5 M 0.016 F 0.001 * 0.017 F 0.005 * 0.027 F 0.002 * 0.018 F 0.002 * 0.021 F 0.001 * 0.031 F 0.002 * 0.044 F 0.004 * 0.13 F 0.003 * DMF 10 7 M 0.024 F 0.001 10 6 M 0.020 F 0.002 10 5 M 0.018 F 0.001
0.032 F 0.003 0.050 F 0.002 0.046 F 0.004 * 0.030 F 0.002 0.074 F 0.002 0.078 F 0.004 0.25 F 0.003 * 0.021 F 0.002 * 0.038 F 0.003 0.024 F 0.006 * 0.030 F 0.002 0.057 F 0.003 0.059 F 0.003 * 0.20 F 0.003 * 0.018 F 0.002 * 0.021 F 0.002 * 0.018 F 0.002 * 0.020 F 0.001 * 0.035 F 0.003 * 0.039 F 0.003 * 0.14 F 0.004 *
Hexane 10 7 M 0.023 F 0.001 10 6 M 0.020 F 0.001 10 5 M 0.017 F 0.002
0.031 F 0.002 0.048 F 0.001 0.044 F 0.002 * 0.034 F 0.002 0.074 F 0.003 0.075 F 0.004 0.25 F 0.003 * 0.018 F 0.002 * 0.027 F 0.002 0.024 F 0.003 * 0.026 F 0.001 0.051 F 0.003 0.071 F 0.004 0.20 F 0.003 * 0.015 F 0.003 * 0.020 F 0.001 * 0.014 F 0.002 * 0.020 F 0.001 * 0.039 F 0.003 * 0.047 F 0.003 * 0.16 F 0.003 *
Values are expressed in nmoles MDA/min/mg protein and represent data F S.E. of 3 experiments. * p < 0.05 when compared to the controls.
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Control
Lymphocyte
Liver
Control
Ethanol
Control
Ethanol
NADPH
CCl4
NADPH
CCl4
NADPH
CCl4
NADPH
CCl4
0.028 F 0.002
0.039 F 0.003
0.058 F 0.001
0.098 F 0.002
0.038 F 0.002
0.080 F 0.001
0.087 F 0.04
0.43 F 0.04
Preimmune IgG 0.3 mg 0.026 F 0.002 0.5 mg 0.025 F 0.002 0.8 mg 0.024 F 0.002
0.036 F 0.001 0.035 F 0.001 0.033 F 0.004
0.055 F 0.003 0.053 F 0.003 0.050 F 0.002
0.090 F 0.001 0.088 F 0.001 0.087 F 0.001
0.036 F 0.002 0.034 F 0.003 0.033 F 0.002
0.076 F 0.002 0.075 F 0.003 0.073 F 0.003
0.081 F 0.003 0.080 F 0.004 0.075 F 0.003
0.39 F 0.03 0.38 F 0.02 0.37 F 0.02
None
Anti-P4502E1 0.3 mg 0.019 F 0.003 0.018 F 0.002 * 0.029 F 0.001 0.033 F 0.001 * 0.024 F 0.003 0.053 F 0.002 0.054 F 0.002 0.23 F 0.02 * 0.5 mg 0.013 F 0.001 * 0.012 F 0.001 * 0.024 F 0.001 0.025 F 0.001 * 0.021 F 0.003 0.038 F 0.002 * 0.050 F 0.002 0.18 F 0.02 * 0.8 mg 0.008 F 0.001 * 0.008 F 0.002 * 0.017 F 0.003 * 0.008 F 0.001 * 0.018 F 0.002 * 0.031 F 0.002 * 0.039 F 0.002 * 0.16 F 0.02 * Values are expressed in nmoles MDA/min/mg protein and represent data F S.E. of 3 experiments. * p < 0.05 when compared to the controls.
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Table 4 Effect of P450 2E1 inhibitors on NADPH and CCl4 supported NADPH dependent lipid peroxidation in whole rat blood lymphocytes and liver microsomes
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observed on in vitro addition of these inhibitors to liver microsomes isolated from control or ethanol pretreated rats (Tables 3 and 4). Similar to that observed with basal lipid peroxidation, DMF, hexane or DMSO produced a significant inhibition of CCl4 supported NADPH dependent lipid peroxidation in the control lymphocytes at relatively higher concentrations of the inhibitors. As observed with basal lipid peroxidation, addition of inhibitors produced a concentration dependent inhibition of CCl4 supported NADPH dependent lipid peroxidation in lymphocytes isolated from ethanol pretreated rats with almost 80–90% inhibition occurring at the higher concentrations. All the solvents were found to be equally effective in inhibiting CCl4 supported NADPH dependent lipid peroxidation (Table 3). Likewise, addition of anti-P450 2E1 produced inhibition of CCl4 supported NADPH dependent lipid peroxidation when added to control or ethanol induced lymphocytes, though the inhibition was much more significant at all the concentrations in blood lymphocytes isolated from ethanol pretreated animals. In vitro addition of preimmune- IgG to the lymphocytes isolated from control or ethanol pretreated rats did not produce any significant change in the basal or CCl4 supported NADPH dependent lipid peroxidation (Table 4). Almost similar pattern of inhibition of CCl4 supported NADPH dependent lipid peroxidation was observed when the inhibitors or Table 5 Effect of P450 2E1 inhibitors on NDMA-d activity in rat liver and blood lymphocytes Lymphocytes
Liver
Control
Ethanol
Control
Ethanol
Sample MeOH
0.98 F 0.04 0.92 F 0.02
2.35 F 0.01 2.29 F 0.06
2.68 F 0.01 2.56 F 0.04
9.38 F 0.03 8.64 F 0.01
DMF 10 3 M 10 2 M 10 1 M
0.85 F 0.02 0.74 F 0.03 0.29 F 0.03 *
1.81 F 0.02 0.96 F 0.02 * 0.54 F 0.01 *
2.04 F 0.02 1.96 F 0.01 1.2 F 0.02 *
6.04 F 0.03 3.23 F 0.02 * 1.9 F 0.02
DMSO 10 3 M 10 2 M 10 1 M
0.78 F 0.02 0.60 F 0.02 0.25 F 0.01 *
0.97 F 0.03 0.68 F 0.02 * 0.51 F 0.02 *
1.78 F 0.01 1.48 F 0.03 0.81 F 0.02 *
3.44 F 0.04 * 2.3 F 0.06 * 1.16 F 0.02 *
Hexane 10 3 M 10 2 M 10 1 M
0.83 F 0.03 0.66 F 0.02 0.37 F 0.02 *
1.16 F 0.04 0.82 F 0.02 * 0.42 F 0.01 *
2.18 F 0.02 2.02 F 0.02 1.28 F 0.03 *
6.2 F 0.01 3.36 F 0.03 * 2.07 F 0.02 *
Preimmune IgG 0.3 mg 0.8 mg
0.80 F 0.01 0.79 F 0.01
2.12 F 0.03 2.06 F 0.02
2.54 F 0.02 2.48 F 0.02
8.42 F 0.01 8.39 F 0.03
Anti P4502E1 0.3 mg 0.8 mg
0.51 F 0.01 0.28 F 0.02 *
0.50 F 0.03 * 0.33 F 0.02 *
1.52 F 0.01 0.68 F 0.01 *
1.76 F 0.02 * 0.42 F 0.01 *
Values are expressed in nmoles HCHO/min/mg protein. * p < 0.05 when compared with control.
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anti-P450 2E1 was added in vitro to the reaction mixture containing liver microsomes isolated from control or ethanol pretreated rats (Tables 3 and 4). Effect of organic inhibitors and anti-P450 2E1 on NDMA-d activity in blood lymphocytes The effect of solvents for eg. DMF, DMSO and hexane and anti-P450 2E1 on the activity of NDMAd, when added in vitro to the lymphocytes isolated from control or ethanol pretreated rats is summarised in Table 5. Addition of DMSO or DMF or hexane or anti-P450 2E1 to the blood lymphocytes isolated from control rats produced a concentration dependent inhibition of the enzyme activity with significant inhibition occuring only at higher concentrations. Similar to that seen in the lymphocytes, significant inhibition of the enzyme activity was observed in liver microsomes with higher concentrations of the inhibitors. Addition of preimmune IgG had no significant effect when added in vitro to liver microsomes or blood lymphocytes isolated from control rats (Table 5). On the other hand, in vitro addition of DMSO or DMF or hexane or anti-P450 2E1 to the blood lymphocytes isolated from ethanol pretreated rats produced a significant concentration dependent inhibition of the enzyme activity with almost 80% inhibition occuring with all the solvents or anti-P450 2E1 at the highest concentrations. Similar pattern of inhibition of the enzyme activity was observed when these inhibitors were added to the liver microsomes isolated from ethanol pretreated rats (Table 5).
Discussion Raucy et al [19] have shown that though considerable variations exist amongst the laboratories in the detection of P450s in blood lymphocytes, it has been shown that P450 2E1 and P450 2D6 exhibit the highest expression and greatest inter-laboratory reproducibility and the concentrations of these P450s in blood lymphocytes may reflect the in vivo activity of the corresponding liver isoenzymes. Even though the physiological role of P450s in blood is highly speculative at present, NADPH dependent (basal) lipid peroxidation and demethylation of N-nitrosodimethylamine in blood lymphocytes have indicated that P450 2E1 expressed in blood lymphocytes are functionally and catalytically active. As observed in the liver, several fold induction in the activity of NDMA-d by pretreatment with ethanol or acetone or pyrazole, have indicated selective enrichment of P450 2E1 isoenzymes. Since higher rates of NADH oxidase activity and NADPH- supported lipid peroxidation occur in liver microsomes of animals treated with P450 2E1 inducers, the enrichment of P450 2E1 in blood lymphocytes following pretreatment with ethanol or pyrazole or acetone may lead to enhanced rate of generation of active oxygen species resulting in increase in the rate of basal lipid peroxidation in peripheral blood lymphocytes [20]. Significant increase in the basal lipid peroxidation in rat blood lymphocytes on in vitro addition of CCl4 have indicated similarities in the NADPH dependent lipid peroxidation in rat blood lymphocytes with the liver. In the liver, carbon tetrachloride has been shown to be homolytically cleaved by P450 2E1 into trichloromethyl radicals and chloride anion which initiate a free radical attack on the membrane lipids leading to peroxidative decomposition of structural lipids [21]. Pretreatment of ethanol, which leads to the selective enrichment of P450 2E1, increases the formation of these radicals from CCl4 and potentiates CCl4 induced liver damage [22]. Similar to that seen in liver microsomes, an increase in NADPH dependent lipid peroxidation was observed on in vitro addition of CCl4 to lymphocytes isolated from control or ethanol pretreated rats. Though the involvement of P450 isoforms other than P450 2E1,
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such as P450 1A2 and P450 2B1 etc., in the CCl4 supported NADPH dependent lipid peroxidation cannot be ruled out in control blood lymphocytes, P450 2E1, which is selectively induced following treatment with ethanol in blood lymphocytes, maybe solely involved to a large extent, in the stimulation of CCl4 supported NADPH lipid peroxidation in blood lymphocytes isolated from ethanol pretreated rats. Similar mechanisms have been shown to occur in liver microsomes isolated from control and ethanol pretreated rats [23]. In vitro studies have also indicated similarities in the catalytic activity of P450 2E1 in blood lymphocytes with the liver isoenzymes. Significant inhibition of NDMA-d activity and NADPHdependent lipid peroxidation on in vitro addition of P450 2E1 inhibitors such as DMSO, DMF or hexane or anti-P450 2E1 to the blood lymphocytes isolated from control rats have indicated that as in liver, P450 2E1 is expressed in control blood lymphocytes and catalyses the activity of NDMA-d and NADPH dependent lipid peroxidation. Similarly, as observed in liver microsomes, greater magnitude of inhibition in the activity of NDMA-d and NADPH dependent lipid peroxidation on addition of organic inhibitors or anti-P450 2E1 to blood lymphocytes isolated from ethanol pretreated animals have indicated enrichment of P450 2E1 in blood lymphocytes following exposure to ethanol. Further evidence indicating similarities in the expression and catalytic activity of P450 2E1 in blood lymphocytes with the liver isoenzyme was provided by significant increase in the magnitude of inhibition of CCl4 supported NADPH dependent lipid peroxidation on in vitro addition of either of DMF, DMSO or hexane or anti-P450 2E1 to lymphocytes isolated from control or ethanol pretreated rats. Even though enzymes other than P450s such as catalase maybe involved in metabolising DMF or DMSO or hexane [24–26] or P450s other P450 2E1 in metabolising CCl4, almost 80–90% inhibition occurring in ethanol induced lymphocytes have further shown that like in liver, CCl4 is cleaved to free radicals by P450 2E1 expressed in blood lymphocytes leading to the initiation of lipid peroxidation. In conclusion, the results of the present study have shown that P450 2E1 expressed in blood lymphocytes catalyses the basal and CCl4 supported NADPH dependent lipid peroxidation and activity of NDMA-d in rat blood lymphocytes. The similarities in the factors regulating and affecting the catalytic activity of P450 2E1 in blood lymphocyte with the liver isoenzyme have further demonstrated that P450 2E1 in blood lymphocytes could be used to monitor and predict the hepatic levels of the enzyme. Furthermore, characterisation of P450 2E1 dependent lipid peroxidation in blood lymphocytes could be of immense significance in identifying intracellular oxidative damage in the alcoholic subjects and the individuals who could be at greater risk to ethanol induced hepatotoxicity. Acknowledgements AD is grateful to CSIR, New Delhi for providing Senior Research Fellowship. The technical assistance of Mr. Kanahiya Lal and Mr. B.S. Pandey and computer assistance of Mr. Mohd. Aslam are gratefully acknowledged. ITRC Communication No. 2201. References [1] Gonzalez FJ. Molecular genetics of the P450 superfamily. Pharmacol Ther 1990;45:1 – 38. [2] Koop DR. Oxidative and reductive metabolism by cytochrome P450 2E1. FASEB J 1992;6:724 – 30.
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