Patent Application: US-4381499-A

Abstract:
the discovery that cyp1b1 protein is detectable in a wide range of human cancers of different histogenetic types , but is not detectable in non - cancerous tissues , gives rise to diagnostic methods for detecting tumors based on this protein as a marker , and to the possibility of tumor therapies involving the protein . a diagnostic method may include the steps of : obtaining from a patient a tissue sample to be tested for the presence of cancer cells ; producing a prepared sample in a sample preparation process ; contacting the prepared sample with an antibody that reacts with human cyp1b1 protein ; and detecting binding of the antibody to cyp1b1 protein in the prepared sample .

Description:
expression of cyp1b1 was investigated in different types of cancers that had developed in a broad range of different anatomical sites ( bladder , breast , colon , kidney , lung , oesophagus , ovary , skin , stomach , uterus , bone and connective tissue , lymph node , brain and testis ). primary malignant tumours of these tissues constitute different histogenetic types ( carcinomas , lymphomas , sarcomas , neuro - epithelial tumours and germ cell tumours ) each with a different biological behaviour . these tumours also represent a range of both common and less common types of cancer . the presence of cyp1b1 was also investigated in a wide variety of normal tissues . immunohistochemistry for cyp1b1 showed that in all the different types of tumour there was strong immunoreactivity for cyp1b1 . cyp1b1 immunoreactivty was localised specifically to tumour cells . non - tumour cells including stromal cells , inflammatory cells , and endothelial cells present in the sections of tumour showed no immunoreactivity for cyp1b1 . there was no significant intra - tumour heterogeneity of cyp1b1 immunoreactivity and only in five out of 133 tumours was cyp1b1 not detected . there was no immunoreactivity for cyp1b1 in any of the normal tissues studied which included liver , kidney , small intestine and lung . the absence or low level of individual forms of p450 in most studies of human cancer ( 15 - 18 ), combined with extrapolation from studies of rodent hepatic carcinogenesis ( 25 ), had led to the general belief that tumour cells do not significantly express p450 . however , we have now shown that cyp1b1 is expressed in a wide variety of malignant tumours of different histogenetic types and is not present in normal tissues , indicating that this p450 is a tumour specific form of p450 . tumours are composed of a variable proportion of tumour cells and non - tumour cells . to identify that a protein is tumour specific , it is important to demonstrate that the protein is localised only to tumour cells . immunohistochemistry allows the direct visualisation of tumour cells and has the spatial resolution to separate tumour cells from non - tumour cells . furthermore , it is important to show there has been no differential degradation of proteins in normal tissue samples compared with tumour samples , and immunoblotting for β - actin ( as a positive control protein ) showed it to be present in every normal and tumour sample indicating there was no protein degradation . in addition , coomassie blue staining of the polyacrylamide gels showed no evidence of protein degradation . moreover , immuno - histochemistry of the tumour samples provides its own internal control as sections of tumour contain non - tumour cells . the presence of cyp1b1 in many types of tumour suggests that this p450 may have a crucial endogenous function in tumour cells and cyp1b1 may contribute to drug resistance that is observed in many types of tumour . cyp1b1 is also likely to be important in tumour development and progression . its identification in a diverse range of cancers of different histogenetic types and is absence from normal tissues appears to make cyp1b1 one of the common changes of a gene product in malignancy ( 26 ). a previous investigation ( 1 ) found mrna in normal tissues . in some tumours , increased ( 2 - 4 ×) mrna was found compared with normal . this might be due to increased transcription mediated by hypoxia inducible factor . this is a novel heterodimeric transcription factor which is induced by hypoxia , and the stimulus in this case may be the hypoxic micro - environment that can exist in tumours , and this factor can have as one of its components the ah receptor nuclear translocator ( 27 ). however , regulation of other forms of p450 is complex ( 2 , 28 ) and the regulation of cyp1b1 in tumours is likely to be complex also , with multiple mechanisms including transcriptional and post - transcriptional factors involved . the tumour - specific expression of cyp1b1 has important consequences for both the diagnosis and treatment of cancer . new diagnostic procedures based on the presence of cyp1b1 in cancer cells can be developed , while the expression of cyp1b1 in tumour cells provides a molecular target for the development of new anti - cancer drugs that are selectively activated by cyp1b1 in tumour cells . since cyp1b1 is found in a wide range of tumours it would be expected that such drugs would be effective in treating many different types of cancer . an important feature is that it would be anticipated these drugs would not be associated with the systematic toxicity that limits the use of current anti - cancer drugs as cyp1b1 is not present in normal tissues especially liver , small intestine and kidney that are the main tissues involved in drug metabolism . thus , a major problem to targeting anti - cancer drugs at tumours based on their selective activation by p450 has been marked hepatic p450 metabolism of drugs resulting in decreased bioavailability and / or undue toxicity . the absence of cyp1b1 protein in liver overcomes this problem . as regards tumour diagnosis , numerous methods for using antibodies to detect a specific protein , including cyp1b1 , in a biological sample are known and can be used in the present invention . any of the various antibody methods can be used alone in practising the present invention . if desired , two or more methods can be used to complement one another . a preferred method for use in the present invention is immunohistochemical analysis . immunohistochemical analysis advantageously avoids a dilution effect when relatively few cancer cells are in the midst of normal cells . an early step in immunohistochemical analysis is tissue fixation , which preserves proteins in place within cells . this prevents substantial mixing of proteins from different cells . as a result , surrounding normal cells do not diminish the detectability of cyp1b1 - containing cancer cells . this is in contrast to assay methods that involve tissue homogenization . upon tissue homogenization , cyp1b1 protein from cancer cells is mixed with proteins from any surrounding normal cells present in the tissue sample . the concentration of cyp1b1 protein is thus reduced in the prepared sample , and it can fall below detectable limits . immunohistochemical analysis has at least three other advantages . first , it requires less tissue than is required by alternative methods such as western blot analysis or immunoassay . second , it provides information on the intracellular localization and distribution of immunoreactive material . third , information on cell morphology can be obtained from the same thin section used to test for the presence of cyp1b1 protein . preferably , when immunohistochemical analysis is employed in the practice of this invention , several thin sections from each tissue sample are prepared and analysed . this increases the chances of finding small tumours . another preferred antibody method for use in the present invention is western blot analysis , ie sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ), followed by immunoblotting . sample preparation for wester blot analysis includes tissue homogenization , and optionally isolation of microsomes . western blot analysis has the advantage of detecting immunoreactivity on proteins that have been separated with high resolution , according to ( apparent ) molecular weight . immunoassays such as antibody capture assays , two - antibody sandwich assays , and antigen capture assays can also be used in the present invention . sample preparation for immunoassays include tissue homogenization , and optionally isolation of microsomes . immunoassays have the advantage of enabling large numbers of samples to be tested relatively quickly , and they offer quantitative precision . principles and practice of immunohistochemistry , western blot analysis , and immunoassays are well known . one of ordinary skill in the art can select suitable protocols and carry out immunohistochemical analysis . western blot analysis , or an immunoassay , in the practice of the present invention ( 30 ). as already noted , the diagnostic aspects of the present invention can conveniently use an antibody that recognizes human cyp1b1 . antibody specificity for cyp1b1 protein is preferable , but not required . preferably , any non - cyp1b1 protein recognized by the antibody is readily distinguished from cyp1b1 , eg , according to apparent molecular weight on a western blot . with selection of an appropriate assay protocol , which is within ordinary skill in the art , the invention can be practised with a polyclonal antibody or a monoclonal antibody . a polyclonal antibody or monoclonal antibody suitable for use in the present invention can be obtained according to conventional procedures ( 30 ). preparation of antibodies that react with cyp1b1 protein is known ( 31 ). procedures for obtaining antibodies that react with human cyp1b1 protein can be carried out using a preparation of non - human cyp1b1 protein , eg , murine cyp1b1 protein . a cyp1b1 protein preparation suitable for eliciting antibodies useful in the present invention can be obtained according to various procedures , including those described ( 31 ). antibodies useful in the present invention can be obtained by immunizing an animal with a preparation containing intact cyp1b1 protein . alternatively , useful antibodies can be obtained by immunizing an animal with a polypeptide or oligopeptide corresponding to one or more epitopes on the cyp1b1 protein . to prepare an antibody according to the latter approach , two 15 - mer peptides corresponding to epitopes on the human cyp1b1 protein were synthesized . each corresponded to a different putative surface loop region of the cyp1b1 enzyme . the first peptide ( designated 217a ) consisted of 14 amino acids , ie , eslrpgaapr dmmd ( seq id no : 1 ). peptide 217a represented amino acid positions 312 - 325 of the deduced amino acid sequence . a carboxy terminal cysteine was included for use in a conjugation reaction . the second peptide ( designated 218a ) consisted of 14 amino acids , ie , ekkaagdshg ggar ( seq id no : 2 ). peptide 218a represented positions 332 - 345 of the deduced amino acid sequence . a carboxy terminal cysteine was added for use in a conjugation reaction . each of these peptides was conjugated directly to klh . male new zealand rabbits were immunized at several anatomical sites using 100 μg of the 217a peptide conjugate or the 218a peptide conjugate . the conjugates were dissolved in 300 μl of pbs mixed with 300 μl of freund &# 39 ; s complete adjuvant . three weeks after the initial immunization , the rabbits were boosted with 50 μg of one of each of the conjugates ( contained in 300 μl pbs mixed with 300 μl of freund &# 39 ; s incomplete adjuvant , injected in several sites ). one week later ( four weeks after the initial injection ), the rabbits were boosted again , using the same protocol . one week after this second boost , the first serum sample was collected . rabbits were subsequently boosted , and serum samples were collected , weekly . serum samples were screened for anti - cyp1b1 titre and specificity by western blotting against a human cyp1b1 - maltose binding fusion protein expressed in e . coli , and human cyp1b1 protein expressed in cos - 1 cells . anti - cyp1b1 igg was purified by immunoaffinity chromatography . the chromatography was carried out using the appropriate cyp1b1 peptide linked directly to a commercial n - hydroxysuccinamide ester of a derivatized , cross - linked agarose gel bead support ( affigel 10 ; biorad , richmond , calif .). conjugation and chromotography were performed according to the vendor &# 39 ; s recommended protocols . in general in the experiments that follow , samples of normal tissue were obtained from tissue specimens that were removed from patients undergoing surgery for malignant disease . normal liver , stomach and small intestine were also obtained from organ transplant donors . all the tissue samples were processed immediately after excision to prevent any degradation of protein or mrna and ensure no deterioration in tissue morphology . we have previously shown that human liver obtained in this way shows no loss or degradation of individual forms of hepatic p450 ( 20 ). tissue blocks for immuno - histochemistry were fixed in 10 % neutral buffered formalin for 24 hours and then embedded in wax , while tissue samples for immunoblotting and mrna analyses were rapidly frozen in liquid nitrogen and stored at − 80 ° c . prior to use . the presence of cyp1b1 protein in tissue samples was investigated using immunohistochemistry ( 21 ) and sodium dodecyl sulphate polyacrylamide gel electrophoresis ( sds - page ) combined with immunoblotting ( 22 ). immunohistochemistry ensures the identification of specific types of cells containing cyp1b1 and is an idea technique for investigating the presence of cyp1b1 in tumour cells as tumours are composed of a variable proportion of tumour cells and non - tumour cells . immunohistochemistry was used to determine the cellular localisation and distribution of cyp1b1 and was performed on formalin fixed wax embedded sections using two antibodies which recognise cyp1b1 . sites of immunoreactivity were detected using an alkaline phosphatase anti - alkaline phosphatase ( apaap ) technique ( 21 ). samples of tumour and normal tissue were fixed in 10 % neutral buffered formalin for 24 hour and then embedded in wax . sections were cut on to glass slides and for immunohistochemistry sections of tumours and normal tissues were dewaxed in xylene , rehydrated in alcohol and then washed sequentially in cold water and 0 . 05m tris - hcl ( ph 7 . 6 ) containing 0 . 15m sodium chloride ( tbs ). the sections were then immunostained with the cyp1b1 antibodies . subsequently , monoclonal mouse anti - rabbit immunoglobulin ( 1 / 100 , dako ltd , high wycombe , bucks ; uk ), rabbit anti - mouse immunoglobulin ( 1 / 100 , dako ) and mouse monoclonal apaap ( 1 / 100 , dako ) were sequentially applied to the tissue sections for 30 minutes each . between antibody applications the sections were washed with tbs to remove unbound antibody . sites of bound alkaline phosphatase were identified using brom - chloro - indolyl phosphate and nitro blue tetrazolium as the enzyme substrate . after incubating the sections for 30 minutes at room temperature , the reaction was stopped by washing the sections in cold tap water . the slides were then air - dried and mounted in glycerine jelly . the sections were examined using bright field light microscopy in order to establish the presence or absence of immunostaining , and its distribution . sds - page and immunoblotting was followed by enhanced chemiluminescenece ( ecl ) technique as described below . immunoblotting was also performed with a monoclonal antibody to β - actin ( clone no . ac - 15 , sigma , poole , dorset , uk ) to show the presence of a positive control protein in tumour and normal samples and indicate that there was no evidence of protein degradation in any of the tissue samples . the presence of cyp1b1 in each tumour that showed cyp1b1 by immunohistochemistry , and the absence of detectable cyp1b1 in normal tissues , was confirmed by western blot analysis . the proteins subjected to western blot analysis were from isolated microsome preparations . microsomes were prepared essentially as described ( 32 ). tissue samples were thawed in 25 ml 0 . 01m tris - hcl buffer , ph 7 . 4 , containing 1 . 15 % kcl before being homogenized in 0 . 01 m tris - hcl buffer , ph 7 . 4 , containing 0 . 25 m sucrose , 15 % glycerol , using an ultra - turrax homogenizer ( type tp 18 / 2 ; janke and kunkel ag , staufen breisgau , germany ). after centrifugation at 15 , 000 × g for 20 min , the supernatant was removed and recentrifuged at 116 , 000 × g for 50 min . the pellet was resuspended a first time in 0 . 1m tris - hcl buffer , ph 7 . 4 , containing 15 % glycerol , 1 mm edta , and recentrifuged at 116 , 000 × g for 50 min . the pellet was resuspended a second time in tris - hcl - glycerol - edta buffer . microsomal protein concentration was determined ( 34 ). a discontinuous polyacrylamide gel system , as described ( 33 ) with modifications ( 32 ), was employed for separation of proteins in the microsomes . 20 μl of a 1 mg / ml preparation of normal samples , and 40 μl of a 0 . 5 mg / ml preparation of tumour samples , in 0 . 125m tris - hcl , ph 6 . 8 , containing 2 . 35 % ( w / v ) sodium dodecyl sulfate , 5 % ( v / v ) 2 - mercaptoethanol , and 0 . 005 % bromophenol blue tracking dye were loaded onto the gel . 10 μl of a 1 mg / ml preparation of human liver microsomes were used as positive controls . samples were run on a 10 % non - gradient gel at 30 ma . following sds - page , resolved proteins were blotted onto a nitrocellulose membrane ( schleicher & amp ; schuell ; dassel , germany ) overnight as described ( 35 ). nonspecific binding sites were blocked with pbs containing 2 % ( w / v ) nonfat milk , 0 . 05 % ( v / v ) tween 20 ™ for 30 minutes at room temperature , with continuous shaking . this buffer was also used for washing stages . the nitrocellulose membrane was then incubated with cyp1b1 - specific antibody ( 1 : 1000 ) for 90 minutes and goat anti - rabbit immunoglobulin horseradish peroxidase conjugate ( 1 : 2000 bio - rad laboratories , hemel hempstead , herts , uk ) for 60 minutes . the membrane was washed for three successive 15 - minute periods and one 60 - minute period after each incubation to remove unbound antibody . bound horseradish peroxidase was then visualized with an enhanced chemiluminescence ( ecl ) kit ( amersham international , aylesbury , bucks , uk ). detection was carried out as described in the ecl protocol , with the x - ray film ( hyperfilm - ecl ; amersham ) being exposed for 30 seconds . nephrectomy specimens ( n = 10 ) excised from primary renal cell carcinoma were used . samples or normal kidney were taken at least several centimetres distant from the edge of each tumour , and only macroscopically viable tumour was sampled . normal human livers ( n = 5 ) were obtained from renal transplant donors and stored at − 80 ° c . prior to use . microsomes of normal kidney , kidney tumours and normal liver were prepared and subjected to the sds - page and immunoblotting procedures in an enhanced chemiluminescence technique ( 21 , 22 ) using an anti - cyp1 polyclonal antibody . recognition of human cyp1b1 was demonstrated using a maltose - binding recombinant cyp1b1 fusion protein expressed in e . coli . expressed cyp1a1 and cyp1a2 were supplied by dr c l crespi , gentest corp , mass ., usa . the results are shown in fig1 : lane 1 human liver , lane 2 expressed recombinant cyp1b1 protein lanes 3 , 5 , 7 , 9 , 11 normal kidney samples , lanes 4 , 6 , 8 , 10 , 12 corresponding kidney tumours . the same amount of microsomal protein ( 30 μg ) was loaded into each lane , thus allowing direct comparison between the kidney and liver samples . as shown in fig1 the kidney tumours and expressed cyp1b1 show a single immunoreactive band at 60 kda corresponding to the molecular weight of expressed cyp1b1 . in normal kidney none of the samples showed an immunoreactive band at 60 kda . in addition , none of the kidney tumours or normal kidney samples showed the presence of cyp1a1 . immunoblotting of liver samples an immunoreactive band at 54 kda corresponding to the molecular weight of cyp1a2 . the intensity of the band at 54 kda showed liver - to - liver variation , whereas there was no cyp1b1 immunoreactive band at 60 kda in any of the liver samples . the expression of cyp1b1 was also investigated in breast cancer using immunoblotting . samples of breast tissue were obtained from patients undergoing surgery either for primary breast cancer or non - neoplastic breast disease . immunoblotting was performed on breast cancers obtained from six patients ( age range 45 - 67 ; three non - smokers , information not available for three patients ), and histologically all these tumours were carcinomas of no special type . the tissue samples were frozen in liquid nitrogen and stored at − 80 ° c . prior to analysis . sds - page and immunoblotting were carried out as described previously . cyp1b1 was detected using the anti - cyp1 polyclonal antibody referred to above . the results are shown in fig2 ; lane 1 human liver , lane 2 expressed cyp1b1 , lanes 3 - 8 breast tumors . as can be seen , a single protein band of molecular weight 60 kda corresponding to the molecular weight of the expressed cyp1b1 protein was identified . as previously , cyp1b1 was not detectable in the liver sample , but cyp1a2 was detected . immunohistochemistry was used to demonstrate the presence of cyp1b1 specifically in a variety of normal and tumour tissues . the results are shown in table 1 and in fig3 . immunohistological localisation of cyp1b1 was investigated in tumours and normal tissues from invasive ductal carcinoma of the breast , endometrial adenocarcinoma , transitional cell carcinoma of the bladder , diffuse high grade malignant lymphoma , high grade astrocytoma of the brain , soft tissue sarcoma ( malignant fibrous histocytoma ), normal liver , normal kidney , normal small intestine . the antibody used was the 218a anti - cyp1b1 polyclonal antibody described above . fig3 shows an immunoblot of cyp1b1 in different types of tumours and normal tissues . lane 1 normal colon , lane 2 colon adenocarcinoma , lane 3 normal kidney , lane 4 carcinoma of kidney , lane 5 normal breast , lane 6 breast cancer , lane 7 normal jejunum , lane 8 normal stomach , lane 9 normal liver , lane 10 malignant mixed müllerian tumour , lane 11 endometrial adenocarcinoma , lane 12 ovarian carcinoma , lane 13 diffuse b cell lymphoma , lane 14 transitional cell carcinoma , lane 15 lung carcinoma , lane 16 positive control ( dioxin - induced achn kidney tumour cells ( panel a only ). in panel b , the same series of tissue samples have been immunoblotted for β - actin , which is present in all normal and tumour samples . a coomassie blue stained polyacrylamide gel of the same series of tissue samples displayed no evidence of protein degradation . the results demonstrated that this p450 is specifically localised to tumour cells , and that there is no cyp1b1 immunoreactivty in normal tissues . reverse transcription polymerase chain reaction ( rt - pcr ) experiments to detect cyp1b1 mrna were carried out as described in mckay et al ( 23 ). rna was extracted from tissue samples and cdna was synthesised from the isolated rna using oligo ( dt ). the cyp1b1 primers had the following sequences : forward 5 ′- aac tct cca tca ggt gag gt - 3 ′ ( nt 2104 - 2123 ); reverse 5 ′- taa gga agt ata cca gaa ggc - 3 ′ ( nt 2573 - 3593 ) giving a pcr product of 489 bp . β - actin was used as a positive control to confirm the presence and integrity of mrna in each sample and the β - actin primers which were brought from stratagene ( cambridge , uk ) had the following sequences : forward 5 ′- tga cgg ggt cac cca cac tgt gcc cat cta - 3 ′ ( nt 1067 - 1105 ); reverse 5 ′- cta gaa gca ttt gcg gtg gac gat gga ggg - 3 ′ ( nt 1876 - 1905 ). pcr with 35 cycles of amplification for both cyp1b1 and β - actin was performed as described ( 23 ). the positive control for cyp1b1 was a 2 . 78 kb cyp1b1 cdna and the negative control was sterile water in place of cdna . after pcr 10 μl of the pcr product was electrophoresed on a 1 . 5 % agarose gel which incorporated 0 . 007 % w / v ethidium bromide and visualized by uv illumination . the cyp1b1 pcr product was sequenced , after purification , by the direct dideoxy sequencing technique with a t7 sequencing kit ( pharmacia , milton keynes , uk ) used according to the manufacturer &# 39 ; s protocol . to further investigate the relative amount of cyp1b1 mrna in normal and tumour tissues , semi - quantitative rt - pcr of normal and tumour kidney samples was performed using serial dilution of cdna ( 24 ). β - actin mrna was used as an internal control ( 29 ). fig4 shows cyp1b1 and β - actin mrna in normal ( a and b ) and corresponding tumour ( c and d ) samples which have been detected by rt - pcr . lane 1 kidney , lane 2 colon , lane 3 skin , lane 4 oesophagus , lane 5 stomach , lane 6 lymph node , lane 7 breast . analysis of the tumours by rt - pcr showed that all tumour samples in which cyp1b1 had been identified contained cyp1b1 mrna . the pcr product was of the expected molecular size when analyzed by agarose gel eletrophoresis . sequencing of the pcr product confirmed identity with cyp1b1 . the absence or low level of individual forms of p450 in most studies of human cancer ( 15 - 18 ), combined with extrapolation from studies of rodent hepatic carcinogenesis ( 25 ), had led to the general belief that tumour cells do not significantly express p450 . however , we have now shown that cyp1b1 is expressed in a wide variety of malignant tumours of different histogenetic types and is not present in normal tissues , indicating that this p450 is a tumour specific form of p450 . tumours are composed of a variable proportion of tumour cells and non - tumour cells . to identify that a protein is tumour specific , it is important to demonstrate that the protein is localised only to tumour cells . immunohistochemistry allows the direct visualisation of tumour cells and has the spatial resolution to separate tumour cells from non - tumour cells . furthermore , it is important to show there has been no differential degradation of proteins in normal tissue samples compared with tumour samples and immunoblotting for β - actin ( as a positive control protein ) showed it to be present in every normal and tumour sample indicating there was no protein degradation . in addition , coomassie blue staining of the polyacrylamide gels showed no evidence of protein degradation . moreover , immunohistochemistry of the tumour samples provides its own internal control as sections of tumour contain non - tumour cells . 1 . t . r . sutter et al ., j . biol . chem . 269 , 10392 ( 1994 ) 2 . s . a . wrighton and j . c . stevens , crit . rev . toxicol . 22 , 1 ( 1992 ) 3 . d . r . nelson et al , pharmacogenetics 6 , 1 ( 1996 ) 4 . t . shimada and f . p . guengerich , chem . res . toxicol . 4 , 391 ( 1991 ); v . nedelcheva and i . gut , xenobiotica 24 , 1151 ( 1994 ); b . k . park , m . pirmohamed , n . r . kitteringham , pharmac . ther . 58 , 385 ( 1995 ). 5 . j . h . capdevila , j . r . falck , r . w . estabrook , faseb j . 6 , 731 ( 1992 ) 7 . e . h . oliw , prog . lipid res . 33 , 329 ( 1994 ) 8 . g . i . murray and m . d . burke , biochem . pharmacol . 50 , 895 ( 1995 ); l . s . kaminsky and m . j . fasco , crit . rev . toxicol . 21 , 407 ( 1992 ); e . g . scheutz et al ., arch . biochem . biophys . 294 , 206 ( 1992 ) 9 . a . k . jaiswal , f . j . gonzalez , d . w . nebert , science 228 , 80 ( 1985 ) 10 . a . k . jaiswal , d . w . nebert , f . j . gonzalez , nucl . acid res . 14 , 6773 ( 1986 ) 11 . d . sesardic , m . pasanen , o . pelkonen , carcinogenesis 11 , 1183 ( 1990 ) 13 . f . j . gonzalez and h . v . gelboin , drug metab . rev . 26 , 165 ( 1994 ); f . p . guengerich , cancer res . 48 , 2946 ( 1988 ); k . kawajiri and y . fujii - 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