Carcinogenesis vol.11 no.7 pp.1183-1188, 1990
Biffffereeitial expression and re; cytoclhrome P45©IIA geee
iom off members off the in tamae tissees
D.Sesardic, M.Pasanen 1 , O.Pelkonen1 and A.R.Boobis2 Department of Clinical Pharmacology, Royal Postgraduate Medical School, London W12 ONN, UK and 'Department of Pharmacology and Toxicology, University of Oulu, SF-90220 Oulu, Finland 2
To whom all correspondence should be addressed
Cigarette smoking increases phenacetin O-deethylase (POD) activity in both the liver and placenta in man, but aryl hydrocarbon (benzo[a]pyrene) hydroxylase (ABH) activity is increased only in the placenta. Whilst there was no correlation between hepatic POD and AHH activities (rs = 0.42, / > >0.1), there was a highly significant correlation between these two activities in placenta (rs = 0.76, / ) 70% of hepatic high-affinity POD activity but had no effect on the placental activity. Furafylline, a methylxanthine that acts as a highly specific inhibitor of P4501A2-dependent activities in man, inhibited all of the high-affinity component of POD activity in human liver, but was at least three orders of magnitude less potent an inhibitor of placental POD and of both hepatic and placental AHH activities. As previously shown in the rat, exposure of man to polycyclic aromatic hydrocarbons, present in cigarette smoke, differentially induces P450IA2 in the liver and P450IA1 in extrahepatic tissues, at least in the placenta. Again, as in the rat, POD activity in the liver is catalysed by P450IA2, but in the placenta of women exposed to polycyclic aromatic hydrocarbons in cigarette smoke POD activity is catalysed by another isoenzyme, most likely P450IA1. Thus, tissuedependent induction and substrate specificity of members of the P450IA family in man, at least in the placenta, appear to be the same as previously shown in the rat.
Introduction The mechanisms responsible for interindividual differences in the pharmacological and toxicological effects of foreign compounds and in susceptibility to chemical carcinogens have been subject to intense research during the past quarter century. It is now generally accepted that differences in the activity of drugmetabolizing enzymes contribute, at least in part, to such variation. Amongst these enzymes, the cytochrome P450 multiisoenzyme system plays a pivotal role. Thus, the relative proportion of individual isoenzymes of cytochrome P450, and as a consequence any alteration in that proportion caused by •Abbreviations: PAH, polycyclic aromatic hydrocarbon; POD, phenacetin O-deethylase; ERDE, 7-ethoxyresorufin O-deethylase; AHH, aryl hydrocarbon fbenzo(a]pyrene) hydroxylase. f
The nomenclature for the isoenzymes of P450 used in this manuscript is that proposed by Nebert el al. (1,2). In this, the two members of the polycyclic hydrocarbon inducible family of P450 are designated P450IA1 and P450IA2, which correspond to P450d and P450c in the earlier nomenclature of Ryan et al. (3). © Oxford University Press
inducers present in the environment, may be a major factor in determining individual susceptibility to adverse drug reactions and chemical carcinogenesis. Those isoenzymes inducible by polycyclic aromatic hydrocarbons (PAHs*), products of the P450IAf genes (1,2), are primarily responsible for the metabolic activation of a number of environmental chemicals, e.g. PAHs (4) and aromatic amines (5), to carcinogenic intermediates. Whereas the phenomenon of induction by PAHs and related compounds is well documented in laboratory animals, and has been recognized in man, the implications that such induction have for tissue specific toxicity remain to be determined. It has long been known that cigarette smoking increases drug metabolism in man by inducing one or more of the enzymes of drug metabolism (6-8). Cigarette smoke comprises at least 1000 different compounds, both organic and inorganic. Some of these are PAHs, and cigarette smoke possesses inducing properties similar to those of PAHs in experimental animals (9,10). Hepatic cytochrome P450 monooxygenase activities that are increased by tobacco smoke in man include phenacetin O-deethylase (POD) an activity that comprises both a high-affinity and a low-affinity component (11); the high-affinity component is catalysed specifically by P450IA2 in both rat (12) and human (13,14) liver. It is this component of POD activity, determined both in vitro (15,16) and in vivo (6,17), that is increased in cigarette smokers, as are 7-ethoxycoumarin O-deethylase ( 1 8 - 2 0 ) and 7-ethoxyresorufin O-deethylase (ERDE) (18) activities. In contrast to the considerable induction of aryl hydrocarbon hydroxylase (AHH) activity that PAHs produce in the rat, in man little or no induction of this activity is observed following exposure to cigarette smoking (18,21). However, in the placentae of women who smoke, both AHH and ERDE activities can be induced by > 100-fold (22,23). Similar observations have been made in women exposed to polychlorinated or polybrominated biphenyls (24). Although constitutive activity in extrahepatic tissues is often lower than that of liver, the dramatic induction that can occur in such tissues, coupled with the relatively low activity of detoxication pathways, has been invoked to explain, in part, the susceptibility of such tissues to chemical carcinogenicity. It has been shown in a number of studies that members of the cytochrome P450IA subfamily exhibit differential inducibility in extrahepatic tissues of the rat (25,26) and rabbit (27), in that whilst both isoenzymes (P450IA1 and P450IA2) are inducible in liver, only P450IA1 is inducible in extrahepatic tissues. We have also shown that whereas POD activity is specifically catalysed by cytochrome P450IA2 in rat liver, this activity is catalysed exclusively by cytochrome P450IA1 in extrahepatic tissues in this species (26,28). Recently, we have shown that in man hepatic POD activity is catalysed by cytochrome P450IA2 and have confirmed the induction of this isoenzyme by cigarette smoking (16). Similarly, Wong et al. (24) have reported the induction of cytochrome P450IA1 in human placenta, following exposure of women to PAHs in the environment. Where placental P450IA1 is induced, 1183
D.Sesardic el al.
it has been suggested that both AHH and ERDE activities are catalysed by this isoenzyme (18,24). In the present study we have investigated whether tissuespecific inducibility of the members of the P450IA subfamily, such as occurs in laboratory species, can also occur in man. Both specific immunological and chemical inhibitors were used to determine the tissue-specific contribution of these isoenzymes to the O-deethylation of phenacetin in man.
Silver Spring, MD) with an excitation wavelength of 396 nm and an emission wavelength of 522 nm. The assay was standardized with authentic 3-hydroxybenzo[«]pyrene, with quinine sulphate as a secondary standard (35). Mono-oxygenase activities of both liver and placenta were analysed under conditions that were linear with respect to time of incubation and protein concentration. The effects of furafylline, a selective inhibitor of human P450IA2-catalysed reactions (13), were determined on both POD and AHH activities of hepatic and placental samples by preincubating the samples with the compound, added in 10 /A methanol to give afinalconcentration of 1 — 100 fiM, at 37°C for 2 min. Activity was then determined as described above.
Materials and methods
Immunological methods The methods for SDS—PAGE and Western blotting of human liver samples have been detailed previously (16). The non-inhibitory monoclonal antibody (MAb) 3/4/2, raised against rat P450c (P450IA1) but recognizing both P450IA1 and P450LA2 in man (30,36,37), was obtained as previously described (38). Immunoinhibition was investigated with a monospecific polyclonal antibody to rat cytochrome P450IA2, which has been designated aP450d(-c) (3). The antibody was generously provided by W.Levin and P.E.Thomas of Hoffmann La Roche, Nutley, NJ. This antibody reacts with only P450IA2 in the rat and cross-reacts with the orthologous isoenzyme in man. Immunoinhibition of metabolic activity was assessed in duplicate samples by incubating purified antibody with microsomal fraction at room temperature for up to 30 min in PBS (10 mM sodium—potassium phosphate buffer, pH 7.4, containing 137 mM sodium chloride and 2,6 mM potassium chloride), using 5-40 mg Ig/nmol total cytochrome P450. Control incubations contained an equivalent amount of preimmune serum.
Chemicals Phenacetin and paracetamol were obtained from BDH Ltd (Poole, UK). [2H3]Paracetamol and all other reagents used in determining POD activity were obtained as previously reported (29). Benzo[a]pyrene and cofactors for metabolic assays (/3-NADPH, |3-NADP + , glucose-6-phosphate and glucose-6-phosphate dehydrogenase, type XV) were all obtained from Sigma Chemical Co. Ltd (Poole, UK). All reagents for SDS-PAGE and for Western blotting were obtained from the sources previously reported (16,30). Furafylline was a generous gift from Laboratorias Almirall (Barcelona, Spain). Human tissues Human liver samples (total n = 11) were from the Human Tissue Bank at the Royal Postgraduate Medical School and via CostNet (Network of European Tissue Banks). All samples were from organ transplant donors. Samples 03005 —03009 were from three female and two male donors, 34.4 ± 19.6 years of age (mean ±SD; range 16—64 years). Two patients had received dexamethasone for a short period, the others were drug-free prior to removal of the liver sample. One sample was from a subject known to have been a cigarette smoker. The smoking status of the other donors was not known. One sample was made available by Dr I.R.Phillips, St Bartholomew's Hospital Medical College, London and the other five samples were from Professor U.A.Meyer, University of Basle, Switzerland. All samples were histologically normal. Human placentae (total n = 11) were obtained after normal delivery at the Department of Gynaecology and Obsterics of Oulu University Central Hospital, Finland. The trophoblastic tissue was separated from coagulated blood and connective tissue and a piece was frozen at —70°C until isolation of the microsomal fraction (31,32). The use of human tissue in these studies was approved by the relevant local research ethics committees and, where appropriate, the coroner. Microsomal fractions were isolated from frozen tissue samples by standard differential ultracentrifijgation, following the method of Boobis et al. (21) for liver and the method of Pelkonen and Pasanen (33) for placenta. Hepatic samples were stored at a protein concentration of 15 — 20 mg/ml and placenta] samples at a protein concentration of 3 0 - 4 0 mg/ml, both in 0.25 M potassium phosphate buffer, pH 7.25, containing 30% (v/v) glycerol. All samples were stored in small aliquots at -80°C until required. Metabolic assays Human hepatic POD activity was determined as described by Sesardic et al. (16), following the method originally published by Murray and Boobis (29). Placental POD activity was determined using 2.0 mg microsomal protein, 20 fiM phenacetin and an NADPH-generating system containing glucose-6-phosphate and its dehydrogenase. Samples were incubated in a final volume of 1 ml containing placental microsomal protein, Tris —HC1 buffer, pH 7.4 (75 mM), magnesium chloride (3 mM), crystalline bovine serum albumin, fraction V (0.7 mg), /3NADP + (2 mM), glucose-6-phosphate (2.5 mM) and glucose-6-phosphate dehydrogenase (diluted to give 1 unit//tl) (3 units), at 37°C for 1 h in a shaking water bath. The reaction was started by the addition of phenacetin in methanol (20 n\), to give a final concentration of 20 ^M. This concentration of methanol had no effect on POD activity. The resultant product, paracetamol, together with its internal standard, deuteroparacetamol, were derivatized with 3,5-bistrifluoromethylbenzoyl chloride, and then analysed by combined gas chromatography/negative ion chemical ionization mass spectrometry on a Finnigan-MAT 4500 system (Finnigan-MAT, San Jose, CA) fitted with a fused silica capillary column (DBS, 30 m x 0.27 mm, J&W Scientific, supplied by Jones Chromatography Ltd, Llanbradach, UK), as described previously (29). Both hepatic and placental AHH activities were assayed by a modification of the method of Atlas et al. (34). The reaction mixture (1 ml) contained Tris-HCl buffer, pH 7.25 (50 mM), magnesium chloride (3 mM), crystalline bovine serum albumin, fraction V (0.7 mg), NADPH (1.2 mM) and microsomal protein (0.2-2.0 mg). Benzo[a]pyrene, in methanol, was added to give a final concentration of 5 nM. Hepatic samples were incubated for 5 min and placenta samples for 25 min at 37°C in a shaking water bath. Fluorescent metabolites were extracted and analysed by spectrofluorometry (Aminco-Bowman, American Instrument Co.,
Other assays Protein concentration was determined by the method of Lowry et al. (39), using crystalline bovine serum albumin, fraction V, as standard. Total hepatic cytochrome P450 content was measured by the method of Omura and Sato (40), assuming an extinction coefficient of 91 mM" 1 cm" 1 between 450 and 480 nm. Cytochrome P450 spectra were recorded on a model 555 split-beam scanning spectrophotometer with microprocessor controlled background correction (PerkinElmer Ltd, Beaconsfield, UK). Analysis of data Results have been expressed as means ± SD or SEM, where appropriate. Statistical analysis was performed by the Mann—Whitney test for unpaired samples using a commercial software package (StatGraphics 2.1, Statistical Graphics Corporation, MD) running on an Olivetti M24 MS-DOS personal computer. The null hypothesis was rejected at P 0.1) (Figure 3A). Eleven full-term placenta! samples were obtained from women whose smoking status was ascertained from their plasma cotinine levels (41). No immunoreactive P450 could be detected in any of these samples either with MAb 3/4/2 or with aP450d(-c). POD and AHH activities were determined in placental samples from both smokers and non-smokers (Table II). Samples from women with the highest plasma cotinine levels (smokers) also had the highest activities for both AHH and POD, presumably due to
80 60 40 20
50 125 [Antibody] (jjg/ml)
induction by cigarette smoke. POD activity of samples from the smokers compared to those from non-smokers was 114 ±143 (n = 5) versus 3.8±2.4 (n = 6) pmol/mg/h (mean ± SD). AHH activity in these samples was 481 ± 425 (n = 5) versus 43 ± 33) (n = 6) pmol/mg/h (Table II). These differences were highly significant (P< 0.025). Mean AHH activity of samples from smokers was 11-fold higher than of those from nonsmokers, and the difference in mean POD activity of these samples was > 30-fold. With placental samples, unlike those from the liver, there was a highly significant correlation between POD and AHH activities (n = 11; rs = 0.76, P