Immunochemical characterization and toxicological significance of P-450HFLb purified from human fetal livers
Mitsukazu Kitada a, Toshiya Kato b, Shigeru Ohmori b, Tetsuya Kamataki c, Koshiro I tahashi ~, F. Peter Guenger ich e, Tadaaki Rikihisa " and Yoshio Kanakubo a
" Hospital Pharmacy, Chiba Unirersity Hospital, Chiba (Japan), I, Faculty of Pharmaceutical Sciences, Chiba Unicersity, Chiba (Japan), " Faculty of Pharmaceutical Sciences, Hokkaido Unicersity, Sapporo (Japan), ,t Kawasaki Seitetsu Hospital, Chiba (Japan)
and " Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt Unit'ersity School of Medicine, Nashcille TN (USA)
Immunochemical properties of P-450HFLb purified from human fetal livers were investigated. P-450HFLb cross-reacted with antibodies to rat P-4501AI but not with antibodies to CYP2A6, CYP2C9, CYP3A7 (P-450HFLa) and rat CYP2BI. In addition, P-450HFLb also cross-reacted with both monospccific antibodies to rat CYPIAI and CYP1A2. However, P-450HFLb was shown to bc an immunochemically distinct form of cytochromc P-450 from P-450PA (human CYP1A2). Immunoblot analysis of human fetal livers with the antibodies to P-450HFLb showed that P-450HFLb was expressed in all fetal livers studied although there appeared to bc individual differences in the amounts of P-450HFLb expressed in fetal livcrs. The formation of mutagcns from IQ (but not from AFBj) in fetal liver homogcnatcs was inhibited by the antibodics to P-450HFLb in a dose dependent manner. These results suggest that P-450HFLb may be a form of human cytochrome P-450 classified into CYP1 gene family, and that the cytochrome P-450 is, in part, responsible for the mutagcnic activation of IQ in human fetal livers as well as CYP3A7 (P-450HFLa).
Introduction
The cytochromes P-450 are the terminal compo- nents of the microsomal multisubstrate monooxygenase system. Based on amino acid sequence identity, liver cytochrome P-450 that constitutes the major xenobiotic metabolizing enzymes is classified into four main gene families [1]. Several lines of evidence have demon- strated interspecies homology of cytochromes P-450 belonging to the same P-450 gene family [2]. The CYP1 gene family comprises two genes, namely CYP1A1 and CYP1A2 [3]. The products of CYPI gene family are designated P-450c and P-450d in the rat [4], P1-450 and P3-450 in the mouse [5], and LM6 and LM4 in the rabbit [6]. Although cytochrome P-450, namely P- 450PA (human CYP1A2) cross-reactive with antibodies
Correspondence to: M. Kitada, Hospital Pharmacy, Chiba University Hospital, 1-8-1 lnohana, Chiba 280, Japan. Abbreviations: SDS-PAGE, sodium dodecylsulfate polyacrylamide gel electrophoresis; AFB~, aflatoxin BI; IQ, 2-amino-3-methylim- idazo(4,5-f)quinoline; Glu P-I, 2-amino-6-methyldipyrido(1,2-a:3',2'- d)imidazole; Trp-P-2, 3-amino-l-methyl-5H-pyrido(4,3-b)indole ac- etate; PAP, peropidase anti-peropidase.
to rat CYP1A2, has been purified and characterized [7], human CYP1A1 has not yet been purified from human liver microsomes. Both CYP1A1 and CYPIA2 have raised much interest, since they are capable of metabolically activating promutagens and carcinogens [8-10]. On the other hand, it is well known that cy- tochrome P-450 is expressed even at early gestational stage in human fetal livers [11] and the pharmacologi- cal and toxicological significance of fetal cytochrome P-450 has been demonstrated [12-13]. Recently, it has been shown that a cytochrome P-450, P-450HFLb, purified from human fetal liver homogenates is active for mutagenic activation of promutagens as well as P-450HFLa (CYP3A7) in a reconstituted monooxygen- ase system [14].
The purpose of this study is to gain a better under- standing of immunochemical properties of P-450HFLb and contribution of the cytochrome to the activation of promutagens in human fetal livers.
Materials and Methods
Materials. A preparative DEAE-5PW and hydroxyl- apatite columns were obtained from Toso, Tokyo, Japan and Koken, Tokyo, Japan, respectively. NADP,
302
glucose 6-phosphate and glucose-6-phosphate dehydro- genase were obtained from Oriental Yeast, Tokyo, Japan. Protein A Sepharose and Sepharose 4B were from Pharmacia, Uppsala, Sweden. Emulgen 911 was kindly provided by Kao-Atlas, Tokyo, Japan. Other chemicals were of the highest grade commercially available.
Purification of cytochrome P-450. CYP2B1 was puri- fied from liver microsomes of phenobarbital-treated rats, and CYP1AI and CYP1A2 were purified from liver microsomes of /3-naphtoflavone-treated rats ac- cording to the methods described previously [15,16]. CYP2A6 and CYP2C9 were purified from microsomes of adult human livers as described previously [17,18]. Purification of P-450HFLa and b from fetal livers was carried out as described elsewhere [14].
Preparation of antibodies. Antibodies against puri- fied cytochrome P-450 were prepared in female New Zealand white rabbits (about 2.5 kg). Rabbits were injected (15-20 sites, subcutaneously) with approx. 20
A Antibodies
CYPIA1 C Y P 2 A 6 CYP2B1
/~g of purified proteins mixed with Freund's complete adjuvant once a week for 2 weeks followed by a booster injection after 2 weeks. Rabbits were bled 4 weeks after the first injection. Since CYPIA1 and CYP1A2 are immunochemically related each to other, CYP1A1 and CYP1A2 antibodies were purified with partially purified preparations of the heterologous proteins, if necessary [19]. The y-globulin fraction of rabbit anti- serum prepared according to the protocol provided by
t h e supplier using protein A Sepharose column was used for immunoinhibition study.
Assay for mutagen-producing actieity. The mutagen- producing activity was measured according to the methods described previously [20]. The induction of umu gene expression induced by the metabolic activa- tion of promutagens was measured using the tester strain Salmonella typhimurium TA1535/pSK1002 car- rying the plasmid pSK1002, which contains a umuC'lacZ-fused gene that produces /3-galactosidase. A typical reaction mixture consisted of 50 mM potas-
Fig. 1. Immunoblot analysis of purified P-450HFLb SDS-PAGE. Immunoblot of purified P-450HFLb and PAP-staining with antibodies to rat CYP1A2, CYP2B1, human CYP2A6, CYP2C9 and CYP3A7 (P-450HFLa) (A), and with monospecific antibodies prepared to rat CYP1A1 and CYP1A2 (B) were performed as described in Materials and Methods. The concentrations of purified P-450HFLb and rat CYP1A1 and CYP1A2
applied were 1.0, 0.45 and 0.10 pmol, respectively.
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1 2 3 4 5 6 St.
Subject number
303
Fig. 2. lmmunoblot analysis of human fetal liver homogenates with antibodies to P-450HFLb. About 10/zg of protein was subjected in each lane, and immunoblot analysis was carried out as described in lhe legend for Fig. 1.
sium phosphate buffer (pH 7.4), an NADPH-gener- ating system (0.33 mM NADP, 8 mM glucose 6-phos- phate, 0.5 unit of glucose-6-phosphate dehydrogenase and 6 mM MgC12), 0.1 mM EDTA, fetal liver ho- mogenates (20 to 40 pmol of cytochrome P-450), 10 /zM promutagen and bacteria in a final volume of 1.0 ml. Promutagens were dissolved in dimethylsulfoxide at a concentration of 4 mM and 2.5 ~1 of the solution was added to the reaction mixture. The mixture was incu- bated aerobically at 37°C for 120 min with shaking. The activity of fl-galactosidase was measured spectrophoto- metrically using o-nitrophenyl-/3-D-galactopyranoside as the substrate. The unit of the enzyme activity was calculated according to the method of Miller [21].
Other methods. The protein concentration was esti- mated by the method of Lowry et al. [22] using bovine serum albumin as the standard. SDS-PAGE and im- munoblotting were performed according to the meth- ods described elsewhere [23,24].
Results
To clarify immunochemical properties of purified P-450HFLb, cross-reactivities of the cytochrome with rat CYP1A1, rat CYP2B1, CYP2A6, CYP2C9 and CYP3A7 (P-450HFLa) antibodies were investigated. As shown in Fig. 1A, purified P-450HFLb cross-re- acted with antibodies to rat CYP1A1 but not with antibodies against rat CYP2B1, CYP2A6, CYP2C9 and CYP3A7 (P-450HFLa). Since rat CYP1A1 and CYP1A2 are immunochemically related each other, purified CYP1A1 and CYP1A2 antibodies were used to gain further information concerning immuno- chemical properties of P-450HFLb (Fig. 1B). The anti- bodies to rat CYP1A1 purified by immunoabsorption was monospecific against rat CYP1A1, and similarly the antibodies to CYP1A2 purified was monospecific against rat CYP1A2. In contrast, P-450HFLb cross-re- acted with both purified antibodies against CYP1A1 and CYP1A2 to a similar extent. On the other hand, P-450HFLb antibodies cross-reacted with P-450HFLb but not with CYP3A7 (P-450HFLa) and rat CYP2B1 (data not shown). Furthermore, as in the case of anti-
bodies to P-450PA (human CYP1A2), no cross-reactiv- ity of P-450HFLb antibodies with purified rat CYP1A1 and CYP1A2 was observed (data not shown).
The antibodies to P-450HFLb recognized a protein having the same molecular weight as the antigen in fetal livers (Fig. 2). Although P-450HFLb was found in all fetal liver samples studied, there appeared to be a large individual difference in the amounts of P- 450HFLb expressed in fetal livers (Table I). The amounts of cytochrome P-450HFLb were estimated to be 0.03 to 0.43 nmol per g of liver, and were much less than those of CYP3A7 (P-450HFLa). The relationship between the amount of P-450HFLb and gestational age was, however, unclear. No correlation was observed between the amounts of P-450HFLb and the activities of benzphetamine N-demethylase, aniline hydroxylase and testosterone 6fl-hydroxylase in fetal livers (data not shown).
Fig. 3 shows the results of immunoblot analysis of adult and fetal livers with P-450HFLb antibodies and / or P-450PA (human CYP1A2) antibodies. Antibodies to P-450HFLb recognized a single protein exhibiting the same mobility on SDS-PAGE as purified P- 450HFLb, in both adult and fetal livers. In contrast, the cross-reactive protein with antibodies to P-450PA (human CYP1A2) was observed only in adult livers but
TABLE I
Amounts o f P-450HFLa and P-450HFLb expressed in human fetal liL'ers
a Values were obtained from previous reports [29]. ND; not deter- mined.
304
P-450PA (human CYPIA2)
J
53k ~ ~
Antibodies
P-450HFLb P-450PA + P-450HFLb
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Antigens Fig. 3. immunochemically cross-reactive proteins with antibodies to P-450HFLb and P-450PA in adult and fetal livers. Immunoblot of adult and fetal livers with antibodies to P-450HFLb and' P-450PA (CYPIA2) was carried out as described in the legend for Fig. 1. After electrophoretically separated proteins were transfered to nitrocellulose sheets, the sheets were treated with antibodies to P-450HFLb a n d / o r P-450PA. The
concentrations of adult liver microsomes and fetal liver homogenates applied were about 2 / zg and 10/xg per lane, respectively.
not in fetal livers, suggesting that P-450PA (human CYP1A2) antibodies do not cross-react with P- 450HFLb. To clarify whether the protein cross-reactive with antibodies to P-450HFLb in adult liver micro- somes is a distinct form of cytochrome P-450 from P-450PA (human CYP1A2), immunoblot and PAP staining was carried out using antibodies raised to both P-450HFLb and P-450PA (human CYP1A2). As ex- pected, two staining bands were observed when adult liver microsomes were used, and only a single staining
tration of IgG added were as specified in the figure.
b~and was found when fetal liver homogenates were used. One of the two staining bands with adult liver microsomes showed the same mobility on SDS-PAGE as P-450HFLb indicating that P-450HFLb is a distinct form from P-450PA (human CYP1A2) in their molecu- lar weights and immunochemical properties, even though P-450HFLb may be classified in CYP1 gene family as well as P-450PA (human CYP1A2).
Effects of antibodies to P-450HFLb on the muta- genic activation of IQ and AFB 1 in human fetal liver homogenates are shown in Fig. 4. The antibodies to P-450HFLb inhibited mutagen-producing activity from IQ by about 50% compared with the value obtained in the absence of the antibodies. In contrast, the muta- genic activation of AFB 1 was virtually unaffected by the antibodies to P-450HFLb. These results indicate that P-450HFLb is responsible for the mutagen forma- tion from IQ in fetal livers. In addition, it is suggested that although P-450HFLb was capable of metabolizing not only IQ but also AFB 1 to mutagens in a reconsti- tuted system [14], contribution of P-450HFLb to muta- genic activation of AFB I may be very little, if any, in fetal livers. These results were in agreement with our previous reports in which CYP3A7 (P-450HFLa) anti- bodies almost completely inhibited the mutagenic acti- vation of AFB I in human fetal livers [13].
Discussion
Cytochrome P-450 comprises a large family of hemoproteins and, within a sub-family, their primary structures have been shown to be well conserved among animal species including human [21]. In human, a
cytochrome P-450, designated P-450PA, purified from adult livers, has been shown to be one of the forms of cytochrome P-450 classified in CYP1 gene family. P- 450PA is cross-reactive with antibodies to P-450d (rat CYP1A2) and is active for mutagen production from promutagens such as 2-amino-anthracene, 2-amino- biphenyl, Glu P-l, Trp P-2 and IQ [22,23]. P-450HFLb contributes to mutagenic activation of IQ, and is also cross-reactive with monospecific antibodies to rat CYP1A1 and CYP1A2, but not with antibodies to other cytochromes P-450 studied. Judging from the immunochemical and catalytical properties of P- 450HFLb, it was suggested that P-450HFLb may also be classified in CYP1 gene family. However, P- 450HFLb appeared to be clearly distinct form of cy- tochrome P-450 from P-450PA (human CYP1A2) as judged by immunoblot analysis of adult and fetal livers.
Antibodies to P-450HFLb recognized a protein ex- hibiting apparently the same mobility on SDS-PAGE as P-450HFLb in adult liver microsomes. We have recently purified cytochrome P-450, designated P-450- HM4, from adult liver microsomes [28]. Although P- 450HFLb was indistinguishable from P-450-HM4 in their molecular weights and immunochemical proper- ties, mutagen-producing activity of P-450HFLb in a reconstituted system was about one order lower than that of P-450-HM4. Therefore, the possibility that both cytochromes P-450 are closely related, but not the same, to each other as in the case of CYP3A7 and CYP3A4, cannot be excluded. Whether the protein cross-reactive with the antibodies in adult liver micro- somes is actually the same form of cytochrome P-450 as P-450HFLb is now under investigation.
Acknowledgement
This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.
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