Gut 1994; 35: 599-603

Cytochrome P450 expression in oesophageal cancer

G I Murray, D Shaw, R J Weaver, J A McKay, SW B Ewen, W T Melvin, M D Burke

AbstractThe cytochrome P450 superfamily of enzymesplay a central part in the metabolism ofcarcinogens and anti-cancer drugs. Theexpression, cellular localisation, and distri-bution of different forms of P450 and thefunctionally associated enzymes epoxidehydrolase and glutathione S-transferases havebeen investigated in oesophageal cancer andnon-neoplastic oesophageal tissue usingimmunohistochemistry. Expression of thedifferent enzymes was confined to epithelialcells in both non-neoplastic samples andtumour samples except that CYP3A was alsoidentified in mast cells and glutathione S-transferase pi was present in chronic inflam-matory cells. CYPlA was present in a smallpercentage of non-neoplastic samples but bothCYP2C and CYP3A were absent. Epoxidehydrolase was present in half of the non-neoplastic samples and the different classes ofglutathione S-transferase were present in a lownumber of samples. In carcinomas CYP1A,CYP3A, epoxide hydrolase, and glutathioneS-transferase pi were expressed in at least 60%of samples. The expression of glutathioneS-transferases alpha and mu were significantlyless in adenocarcinoma compared withsquamous carcinoma.(Gut 1994; 35: 599-603)

metabolism of compounds initially metabolisedby P450. Epoxide hydrolases participating inxenobiotic metabolism comprise mainly onemicrosomal and one cytosolic form, which con-vert potentially toxic epoxides to less reactivedihydrodiols, although subsequent reoxidationcan lead to the formation of diol-epoxide deriva-tives, which are often more toxic than the parentcompounds.5 The glutathione S-transferases,like the cytochromes P450, are a complex genefamily of enzymes, which catalyse the conjuga-tion of reduced glutathione with a variety ofelectrophilic compounds generally resulting inless toxic, more hydrophilic compounds, whichcan be more easily excreted.6'5 Like the cyto-chromes P450 the liver is the main organ inwhich the glutathione S-transferases are expres-sed and specific classes of glutathione S-trans-ferases are present in many extrahepatic tissues.There is little information regarding the

presence, distribution, and localisation ofdifferent xenobiotic metabolising enzymes ineither normal oesophagus or oesophagealtumours and in this study we have investigatedthe expression of different forms of P450,epoxide hydrolase, and glutathione S-transferasein oesophageal carcinoma and non-neoplasticoesophageal tissue.

Materials and methods

Departments ofPathologyG I MurrayD ShawJ A McKayS W B Ewen

Biomedical SciencesR J WeaverM D Burke

and Molecular and CellBiologyW T Melvin

University of Aberdeen,AberdeenCorrespondence to:Dr G I Murray, Department ofPathology, University ofAberdeen, Foresterhill,Aberdeen AB9 2ZD.Accepted for publication20 September 1993

Oesophageal cancer is one of the commonestmalignancies of the alimentary tract and thistumour is often intrinsically resistant to anti-cancer drugs.' The geographical distribution ofoesophageal cancer has led to the hypothesis thatexposure to various toxic environmental ordietary chemicals is an important aetiologicalfactor.2 The principal groups of enzymesresponsible for metabolising toxic exogenouschemicals, including anti-cancer drugs andcarcinogens, are the cytochromes P450, epoxidehydrolases, and glutathione S-transferases.317Therefore these functionally associated groupsof enzymes have important roles in determiningthe susceptibility of tissues to the toxic effects ofxenobiotics and the response of tumours to anti-cancer drugs.4 10The cytochromes P450 are a multigene

superfamily of haem containing enzymes thatmetabolise both xenobiotics (including carcino-gens, mutagens, and therapeutic drugs) and avariety of endogenous compounds (includingsteroid hormones, arachidonic acid, and fattyacids).4"'4' The major xenobiotic metabolisingfamilies of P450 are primarily expressed in theliver, although specific forms of P450 are presentin particular extrahepatic tissues. The epoxidehydrolases and glutathione S-transferase are twogroups of enzymes that participate in the further

ANTIBODIESCYPlA and CYP3A were identified using mono-clonal antibodies RM3'6 and HL3'78 respect-ively, which have been raised and characterisedin our laboratories as described previously.(Individual families and forms ofP450 are identi-fied in this study using the designation CYPaccording to the recent updated nomenclaturefor cytochrome P450.'2 The P450 forms recog-nised by immunoreactivity in this study areclassified according to their sub-family and notidentified as being individual members of sub-families as it is not known whether the antibodiesare specific to certain sub-family members).CYP2C was identified with a rabbit polyclonalantibody raised in our laboratories againstthe purified human hepatic form CYP2C9,P45OhB. 19 Epoxide hydrolase was recognisedusing a rabbit polyclonal antibody raised andcharacterised in our laboratories against a puri-fied preparation of human hepatic microsomalepoxide hydrolase.20 Three major cytoplasmicclasses of glutathione S-transferases (alpha, mu,and pi) were identified individually using rabbitpolyclonal antibodies obtained from NovocastraLaboratories, Newcastle upon Tyne. All theantibodies used in this study recognise antigenicepitopes, which are resistant to formalin fixationand wax embedding.

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Murray, Shaw, Weaver, McKay, Ewen, Melvin, Burke

TISSUESpecimens of oesophageal cancer and non-neoplastic oesophagus were obtained fromoesophagectomy specimens submitted to theDepartment of Pathology, University ofAberdeen for diagnostic purposes. All the tissuesamples had been fixed in 10% neutral bufferedformalin for 24 hours at room temperature andthen routinely processed to paraffin wax.

IMMUNOHISTOCHEMISTRY2'Formalin fixed wax embedded sections (4 [tm)of oesophagus were dewaxed in xylene, rehy-drated in alcohol, and then washed sequentiallyin cold water and 0-05 M TRIS-HCl (pH 7-6)containing 0415 M sodium chloride (TBS) andthen immunostained with various antibodies asdescribed below. Both negative and positivecontrols were incorporated in the immunohisto-chemical procedure. Negative controls used inplace of the primary antibody were either TBS(control for monoclonal antibodies), or normalrabbit immunoglobulins (control for polyclonalantibodies). Positive controls were sections ofnormal formalin fixed wax embedded humanliver as liver is known to express all the enzymesbeing studied.The sections were examined using transmitted

light microscopy to establish qualitatively thepresence or absence of immunostaining, and itsdistribution.

Correlations between the expression of thedifferent enzymes were determined using the x2test with Yates's correction.

MONOCLONAL ANTIBODIESSections of tissue were incubated for one hourwith each of the monoclonal antibodies at thefollowing dilutions: anti-CYPlA (RM3), 1/20

TABLE I Number (percentage) ofoesophageal samplesexpressing different number ofxenobiotic metabolisingenzymes

No ofxenobioticmetabolising Non-neoplastic Squamousenzymes oesophagus carcinoma Adenocarcinomaexpressed (n=-O) (n=25) (n=25)0 2 (20) 0 01 4 (40) 2(8) 2(8)2 1(10) 4(16) 5 (20)3 1(10) 0 8 (32)4 1(10) 2(8) 1(4)5 1(10) 8 (32) 5 (20)6 0 6 (24) 3(12)7 0 3 (12) 1(4)

TABLE II Number (percentage) ofnon-neoplastic oesophageal samples and distinct histologicaltypes ofoesophageal cancer expressing different xenobiotic metabolising enzymes

Non-neoplastic Squamousoesophagus carcinomta Adenocarcinoma

Xenobiotic metabolising enzyme (n= 10) (n=25) (n=25)

CYPIA 3 (30) 17 (68) 15 (60)CYP2C 0 8 (32) 5 (20)CYP3A 0 19(76) 17 (68)Epoxide hydrolase 5 (50) 21(84) 24 (96)Glutathione S-transferase alpha 4 (40) 19(76) 8 (32)Glutathione S-transferase mu 2 (20) 15 (60) 5 (20)Glutathione S-transferase pi 2 (20) 16 (64) 16 (64)

dilution in TBS of a 50% ammonium sulphateprecipitate of hybridoma culture supernatant,and anti-CYP3A (HL3) as undiluted hybridomaculture supernatant. Sites of antibody bindingwere shown using an alkaline phosphatase anti-alkaline phosphatase (APAAP) technique.Rabbit antimouse immunoglobulin (1/100 con-taining 1% normal human serum, Dako Ltd,High Wycombe, Bucks) and monoclonalAPAAP (1/100, Dako) were sequentially appliedto the tissue sections for 30 minutes each.Between antibody applications the sections werewashed with TBS to remove unbound antibody.Sites of bound alkaline phosphatase were shownusing an enzyme substrate solution containing3 mg bromo-chloro-indolyl phosphate (SigmaChemical Co Ltd, Poole, Dorset), 10 mg nitroblue tetrazolium (Sigma), 6 mg sodium azide,and 4 mg levamisole (Sigma) in 10 ml 0 05 MTRIS-HCl buffer (pH 9 0) containing 0-2%magnesium chloride. After incubating thesections for 30 minutes at room temperature, thereaction was stopped by washing the sections incold tap water. The slides were then air dried andmounted in glycerine jelly.

POLYCLONAL ANTIBODIESAnti-CYP2C was applied to tissue sections at adilution of 1 in 500 (stock solution 20 mg/ml inTBS prepared from the lyophilate of a 50%ammonium sulphate precipitate of immunisedrabbit serum). Anti-epoxide hydrolase wasapplied at a dilution of 1/50 of a stock 20 mg/mlsolution, prepared from the lyophilate of a 50%ammonium sulphate precipitate of immunisedrabbit serum. Antibodies to the different classesof glutathione S-transferase were applied at thefollowing dilutions of a stock solution (proteinconcentration 1 mg/ml): glutathione S-trans-ferase alpha 1/100; glutathione S-transferase mu1/50; glutathione S-transferase pi 1/200. Theantibodies were applied to tissue sections for onehour at room temperature, and the sites ofboundantibody shown using the APAAP technique.After removal ofunbound primary antibody, thesections were incubated with monoclonal mouseantirabbit immunoglobulin (1/100, Dako) for30 minutes before application of rabbit anti-mouse immunoglobulin, APAAP, and alkalinephosphatase substrate solution. The sectionswere then processed as described above.

Results

NON-NEOPLASTIC OESOPHAGUSTissue samples obtained from proximal resec-tion margins of 10 oesophagectomy specimensthat had been excised for squamous carcinomawere used to investigate the presence of thevarious enzymes in non-neoplastic oesophagus.These specimens contained all layers of theoesophagus including both squamous epitheliallining and muscularis. CYPIA immunoreact-ivity was identified in 30% of samples and therewas no immunoreactivity for either CYP2C9 orCYP3A in any of the non-neoplastic specimensof oesophagus. Epoxide hydrolase was identifiedin 50% of the samples and immunoreactivity for

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Cytochrome P450 expression in oesophageal cancer

the different classes of glutathione S-transferasealpha, pi, and mu was present in 40, 20, and 20%of the samples respectively. In all cases thepositive immunoreactivity was identifiedwith the stratified squamous epithelium withimmunostaining being present in all layers of theepithelium. Connective tissue, blood vessels,and smooth muscle showed no immunostrain-ing.

OESOPHAGEAL CANCERFifty oesophageal cancers were studied consist-ing of 25 squamous carcinomas and 25 adeno-carcinomas. Based on immunoreactivity, all thecarcinomas expressed at least one enzyme andfour tumours expressed all seven enzymes (TableI). CYP1A, CYP2C, and CYP3A were identifiedin 32, 13, and 36 tumours respectively (64, 26,and 72% of the neoplasms respectively). Therewas widespread expression of epoxide hydrolasewith 45 (90%) of tumours showing positiveimmunoreactivity. Glutathione S-transferasealpha, mu, and pi were present in 27, 20, and 32of carcinomas respectively (54, 40, and 64% oftumours respectively). Table II summarises thedetailed expression of the various xenobioticmetabolising enzymes in the different histo-logical types of oesophageal cancer. The expres-sion of glutathione S-transferase alpha (x2,p=0005, 1 degree of freedom) and glutathioneS-transferase mu (x2, p=0 009, 1 degree offreedom) were significantly less in adeno-carcinomas compared with squamous carcino-mas. The expression of all the other enzymes wassimilar in the different histological types ofoesophageal cancer.

Immunoreactivity for each enzyme waspresent in the cytoplasm of tumour cells (Figs1-3), although in a few cases nuclear staining forthe different classes of glutathione S-transferases

Figure 2: Immunoreactivityfor epoxide hydrolase is identifiedwithin tumour cells ofan adenocarcinoma ofthe oesophagus(original magnification x 160).

was also seen. The immunoreactivity for eachenzyme had a uniform distribution throughouteach tumour. In addition, mast cells in normaland carcinoma samples consistently showedstrong positive staining for CYP3A whileglutathione S-transferase pi immunoreactivitywas also identified in chronic inflammatory cells.

DiscussionWe have investigatedspecific sub-families

the expression of threeof cytochrome P450

Figure 1: All the tumour cells ofa moderatelv differentiatedsquamous carcinoma ofoesophagus show CYPIAimmunoreactivt4y (original magnification x 160).

Figure 3: Expression ofglutathione S-transferase alpha in anoesophageal squamous carcinoma. All the tumour cells showstrong immunoreactivity (original magnification x 160).

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602 Murray, Shaw, Weaver, McKay, Ewen, Melvin, Burke

(CYP1A, CYP2C, CYP3A) and the functionallyassociated enzymes epoxide hydrolase andglutathione S-transferase in oesophageal cancerand non-neoplastic oesophagus. The presence inthe diet of carcinogens or pro-carcinogens,which can be metabolically activated is animportant factor in the development of oesopha-geal cancer2 and these compounds are generallymetabolised by the xenobiotic metabolisingforms of cytochrome P450. The outcome ofmetabolism in terms of activation (toxicity) ordeactivation (detoxification) will depend on thecellular localisation, comparative amount, andactivity of the different xenobiotic metabolisingenzymes present in a particular tissue. In non-neoplastic oesophagus there was only a lowfrequency of expression of the enzymes studiedand in particular there was no expression ofCYP3A. Samples of non-neoplastic oesophaguswere obtained from the proximal resectionmargins of oesophagectomy specimens that con-tained tumour. Although these specimens dis-played no histological abnormality they may notbe functionally strictly normal, as they had beenobtained from specimens that contained tumour.Ethically, however, it is not possible to obtainentirely normal oesophagus. A low level of P450associated mono-oxygenase activity towards N-nitrosomethyl-n-amylamine has been identifiedin microsomes prepared from non-neoplastichuman oesophageal mucosa although theparticular form or forms of P450 contributing tothis activity were not identified.22

Within the human alimentary tract CYP3A ispresent in a high concentration in the smallintestine'8 23 but either absent or present at a lowconcentration from colon2' and it is suggestedthat a high concentration of CYP3A in the smallintestine protects the small intestine from thedevelopment of cancer.24 Conversely, theabsence of CYP3A from other sites in thealimentary tract may contribute to the frequentdevelopment of malignancy. The failureto detect immunoreactive CYP3A in non-neoplastic oesophagus may support thishypothesis.

There have been no extensive investigations ofdrug metabolising enzymes in oesophagealneoplasia. The only xenobiotic metabolisingenzyme that has been studied in oesophagealmalignancy is the pi class of glutathione S-transferase.2127 Both glutathione S-transferase pimRNA and protein are present in low amountsin non-neoplastic oesophagus but at an increasedlevel in a high proportion of oesophagealsquamous carcinomas.The two different histological groups of oeso-

phageal tumours, namely squamous carcinomaand adenocarcinoma, generally showed a similarpattern of expression of the different enzymesstudied with the exception that the glutathioneS-transferases alpha and mu were expressedsignificantly less in adenocarcinoma thansquamous carcinoma. The differential expres-sion of these forms of glutathione S-transferasemay provide an opportunity clinically for theimproved selection of anti-cancer drugs based onknowledge of their metabolism by these forms ofglutathione S-transferases.

Almost all the tumours displayed positive

epoxide hydrolase immunoreactivity and wehave previously identified epoxide hydrolaseimmunoreactivity in a variety of different malig-nant tumours including hepatoma,20 coloncancer, and breast cancer,28 which suggests thatepoxide hydrolase expression may be a commonmolecular event in malignancy.

In conclusion, we have found non-neoplasticoesophageal epithelium to be characterised bya low frequency of expression of differentxenobiotic metabolising enzymes, with CYP2Cand CYP3A being undetectable. In oesophagealcancer the phenotypic expression of the differentenzymes was complex with the presence in atleast 60% of all tumours ofCYPlA and CYP3A,epoxide hydrolase and glutathione S-transferasepi. The frequent expression of the differentxenobiotic metabolising enzymes in oesophagealcancer may contribute to the anti-cancer drugresistance that is characteristic of oesophagealcancer.

This work has been funded by grants from the Scottish OfficeHome and Health Department (K/MRS/50/C1699), AberdeenRoyal Hospital NHS Trust, and the Wellcome Foundation Ltd.

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