Introduction

Abstract Introduction Results Discussion References

Ah receptor ligands, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD¹), planar polychlorinated biphenyls (PCBs) and 3-methylcholanthrene induce enzymes in the CYP1A subfamily, CYP1A1 and CYP1A2, in rodents and other mammals (1). These enzymes exhibit catalytic differences. For example, 7-ethoxyresorufin deethylase (EROD) activity is commonly used as an index of CYP1A1 activity in rat liver because it has been found to be catalyzed principally by CYP1A1 with only about 20% of the activity being catalyzed by CYP1A2 (2). CYP1A2 is preferentially active in different activities, including the 2-hydroxylation of estradiol (3).

Recently the oxidation of uroporphyrinogen to uroporphyrin (UROX) was shown to be preferentially catalyzed by rat and mouse CYP1A2 rather than 1A1 (2, 4). Uroporphyrinogen, an intermediate in heme biosynthesis, is normally converted to coproporphyrinogen by the enzyme, uroporphyrinogen decarboxylase (5). The oxidation of uroporphyrinogen prevents its conversion to coproporphyrinogen and results in accumulation of uroporphyrin. UROX activity is increased by inducers of the CYP1A subfamily in rodents and is considered to be an essential participant in the massive accumulation of hepatic uroporphyrin (URO) caused by these compounds (2, 5). Ah receptor ligands induce UROX in chick embryo hepatocytes (2, 6, 7), but the question of which CYP enzymes are involved has not been clarified.

In contrast to rodent liver, previous investigations indicated that in chicken liver there was only one P450 form in the 1A subfamily induced by 3-methylcholanthrene and similar compounds (2). This one form appeared to catalyze both EROD and UROX activities, based on the effects of an antibody prepared with a single 3-methylcholanthrene-induced form (8). Two TCDD-induced CYP enzymes from chick embryo liver have recently been identified and purified (9-11). One enzyme, which has been designated CYP1A4, was found to be responsible for EROD and aryl hydrocarbon hydroxylase (AHH) in chick embryo. The other CYP enzyme, CYP1A5, was found to be responsible for TCDD-induced arachidonic acid epoxygenation and the 4-hydroxylation of tamoxifen in chick embryo liver (10, 12) but was inactive in EROD or AHH activities (9,11). The cDNA-derived amino acid sequences of each of these chick CYP enzymes permit both to be classified in the CYP1A subfamily (13), but the sequences of both are more similar to CYP1A1 than to 1A2 and neither can be said to be the direct ortholog of CYP1A1 or 1A2 (13).

To better understand the function of CYP1A enzymes in nature, we therefore examined the abilities of the two avian CYP1A enzymes to carry out UROX, an activity preferentially catalyzed by mammalian CYP1A2. We examined the abilities of each of the TCDD-induced chick CYP enzymes to catalyze UROX in reconstitution assays and the effect of TCB in stimulating reconstituted UROX activity. We also investigated the dose-response relationships for the induction of UROX and EROD in chick embryo liver microsomes and cultured hepatocytes and compared the induction of UROX and EROD in chick embryo liver and kidney.

Materials and Methods

Materials. Sources of chemicals were as follows: 3-methylcholanthrene, ketoconazole and dilaurylphosphatidylcholine, Sigma Chemical (St Louis, MO); 3,4,3,4-tetrachlorobiphenyl (TCB) and 3,4,5,3,4,5-hexachlorobiphenyl, Ultrascientific (North Kingstown, RI); dimethyl sulfoxide, Fluka (Hauppage, NY); uroporphyrin, Porphyrin Products (Logan, UT). The CYP enzymes, CYP1A5 and CYP1A4, were purified from the livers of TCDD-treated embryos as described previously (9, 11). Rat liver NADPH-cytochrome P450 reductase used for the reconstitution studies was purified as described (9). Recombinant rat liver NADPH-cytochrome P450 reductase used for microsome supplementation was a gift from Drs. C. Fisher and R. Estabrook, University of Texas Southwestern Medical Center, Dallas, TX.

Chick Embryo Hepatocyte Cultures. Cultures were prepared as described previously (14). Microsomes were prepared by centrifuging sonicates of the cells at 10,000g for 10 min, and then the superntatant was centrifuged at 100,000g for 60 min. The pellet was resuspended in 0.25 M sucrose/0.05 M HEPES/1 mM EDTA, pH 7.4 and stored at 60^o. There was no loss of activity for up to 6 months.

Treatment of Animals. Twelve-day-old chickens were treated by intraperitoneal injection with 3-methylcholanthrene (50 mg/kg) in corn oil (20 mg/ml). After 48 hr, chickens were killed by decapitation and liver and kidney microsomes were prepared by differential centrifugation as described (4). Sixteen-day-old chick embryos were treated with increasing doses of TCB in 0.012 ml acetone pipetted through a hole in the shell onto the air sac or were treated with 3-methylcholanthrene (0.5 mg/egg) in dimethyl sulfoxide (0.1 ml) injected into the embryo. After 48 hr, liver microsomes were prepared and stored as previously described (4). Each set of data presented reflect experiments performed at least twice.

Uroporphyrinogen Oxidation (UROX) by Microsomes or Purified P450s. UROX activity was monitored by appearance of uroporphyrin fluorescence at room temperature in a Perkin-Elmer 650-10S spectrophotofluorimeter (excitation wavelength, 400 nm; emission wavelength, 618 nm) with excitation and emission slit widths at 3.5 nm and 20 nm, respectively (2). Uroporphyrin I was used as a standard. The assay mixture contained 0.21 ml of assay buffer (0.25 M sucrose/0.05 M HEPES/1 mM EDTA, pH 7.4), 0.032 ml of a microsomal suspension or reconstitution complex containing 32 pmoles of cytochrome P450, 0.005 ml of a NADPH-generating system (125 mg of sodium isocitrate, 40 mg of nicotinamide, 35 mg of NADPH, 70 mg of MgCl₂0.6H₂O, and 0.2 ml of isocitrate dehydrogenase (10 units) dissolved in 0.75 ml of assay buffer) and TCB at 7 -14 µM, in a total volume of 0.25 ml. The reconstitution complex contained in addition to the purified CYP1A5 or CYP1A4, L-dilaurylphosphatidylcholine at 4-16 µg/ml and NADPH P450-reductase as described (11). The ratio of P450 to reductase was 1:1.5. TCB was routinely included in the UROX assay because it has been found to markedly stimulate UROX activity (6, 7) as shown in fig. 1. Activation has been shown to be maximal at 3 µM, but concentrations up to 20 µM are not inhibitory. In contrast, EROD activity does not require the addition of TCB and in fact is inhibited by it². Uroporphyrinogen I was freshly made from uroporphyrin I by reduction with sodium amalgam and dilution into buffer containing 40 mM DTT (2). The reaction was initiated by the addition of 0.003 ml uroporphyrinogen to give a final concentration of 5 µM. The rate of uroporphyrin fluorescence was measured for up to 5 min.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Stimulation of UROX activity in chick embyro hepatic microsomes by TCB. Hepatic microsomes were prepared from chick embryos treated with 3-methylcholanthrene as described in Materials and Methods. Clear bars, no TCB; shaded bars, 7 µM TCB. Values are means and ranges for duplicate determinations of microsomes prepared from pools of six livers.

Ethoxyresorufin deethylase (EROD). Microsomal EROD activity was measured fluorimetrically (excitation wavelength, 530 nm; emission wavelength, 590 nm; slit widths, 10 nm). The assay mixture was essentially the same as in the UROX assay, except for the addition of 1 mg bovine serum albumin/ml (final concentration) (2). The reaction was started by the addition of 0.003 ml of 7-ethoxyresorufin in DMSO to give a final concentration of 1 µM, and the fluorescence was measured for 2-3 min. Resorufin was used as a standard. The data for reconstituted EROD presented in fig. 2 were determined previously (11) using the same purified P450 preparations used in the present study.

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Reconstitution of uroporphyrinogen oxidation (UROX) (A) and 7-ethoxyresorufin O-deethylase (EROD) (B) catalyzed by purified chicken CYP1A5 and CYP1A4. Details of the assays for reconstituted purified CYP enzymes are described in Materials and Methods. For UROX, the reaction mixture contained 7 µM TCB. The values for reconstituted EROD were determined previously (11) using the same purified P450 preparations used in the present study. Values are means and ranges for duplicate determinations.

Other Assays. Uroporphyrin in cultured cells was measured spectrofluorimetrically (15). Protein concentrations were determined by the method of Lowry et al. (16), using bovine serum albumin as a standard. Cytochrome P450 concentrations were determined by the method of Omura and Sato (17).

	Results

Abstract Introduction Results Discussion References

Stimulation of UROX Activity in Chick Embryo Liver Microsomes. Fig. 1 shows that treatment of chick embryos with the Ah receptor ligand, 3-methylcholanthrene, increased liver microsomal UROX activity 3-fold as compared with the untreated controls. Inclusion of TCB in the reaction mixtures had no effect on the UROX activity of the control microsomes but stimulated UROX by 8-fold in microsomes from 3-methylcholanthrene-treated chick embryos. The degree of UROX induction and the effects of TCB are similar to results previously reported (2, 6, 7). The mechanism of the enhancement of induced UROX by TCB is not understood (18).

UROX Activity of Purified Chick CYP1A5 and CYP1A4. Fig. 2 compares the ability of the two purified chick enzymes to carry out UROX activity in reconstitution assays. CYP1A5 was eight times more active than CYP1A4 in catalyzing UROX activity. In contrast, the EROD activity of the same preparation of CYP1A5 was less than 5% of the activity of CYP1A4 (11).

Dose Responses for Induction of UROX and EROD in Chick Embryo Liver and Cultured Chick Hepatocytes. Treatment of chick embryos in ovo with TCB produced almost identical dose-dependent increases in hepatic microsomal UROX and EROD (r²= 0.98) (fig. 3A). In other experiments using 3-methylcholanthrene as an inducer, nearly superimposable dose response relationships for UROX and EROD were also found (data not shown).

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Dose responses for effects of planar halogenated biphenyls on induction of UROX and EROD in livers of 15-day-old chick embryos (A) and in hepatocyte cultures (B) prepared from 15-day-old chick embryos. A. Chick embryo liver microsomes: Increasing doses of 3,4,34-tetrachlorobiphenyl (TCB) were administered in 0.012 ml acetone on the air sac of each embryo. After 18 hr, liver microsomes were prepared. Each point represents the mean of duplicate assays of pools of 5-6 livers with the range indicated by a vertical bar. Where no bar is indicated the range fell within the symbol. () UROX; () EROD. B. Chick hepatocyte cultures: Cultures were prepared in 10-cm dishes as described in Methods. Cultures were treated on the second day of the culture with increasing concentrations of 3,4,5,3,4,5-hexachlorobiphenyl (HCB) for 18 hr. Cells from three dishes were pooled for each HCB concentration. The 100,000g membrane fractions were prepared and assayed for UROX and EROD activities as described in Materials and Methods. UROX activity in untreated cells was 4.6 pmol URO/min/mg protein and this value has been subtracted from the values obtained for the treated cells. () UROX, () EROD.

Effect of Added NADPH Cytochrome P450 Reductase on Microsomal UROX Activity. It was shown previously that in sonicates of cultured hepatocytes the addition of NADPH cytochrome P450 reductase increased CYP1A5 mediated arachidonic acid metabolism 11-fold, but EROD activity only by 20% (19) and that the addition of extra reductase to microsomes increased tamoxifen hydroxylation about 3-fold (12). To exclude the possibility that the results of the dose-response experiments were being affected by limitations in reductase, we examined the effects of added reductase on UROX and EROD activities in liver microsomes of 3-methylcholanthrene-treated chick embryos. Only small effects, an increase of 40% in UROX and a decrease of 20% in EROD, were observed, indicating that UROX activity, unlike some other CYP1A5-mediated activities, was not much affected by extra reductase. In addition, the limitations in reductase activity did not affect the dose-response results.

Induction of UROX and EROD in Chicken Liver and Kidney. Previous studies showed that CYP1A5 and CYP1A4 were both increased in liver and kidney by Ah receptor ligands. The capacity of 3-methylcholanthrene to induce UROX activity in kidney of 12-day old chickens was also examined. Table 2 shows that treatment of chickens with 3-methylcholanthrene increased UROX and EROD in kidney as well as in liver. The activities and the degree of increase were lower in kidney than liver, as previously observed for CYP1A5 and CYP1A4 in chick embryo kidney (11). The ratio of induced EROD:UROX was 21:1 in liver and 8:1 in kidney, indicating that the relative proportions of CYP1A4 and CYP1A5 differ in liver and kidney and that there was relatively more CYP1A5 in kidney than in liver. The low EROD activity in kidney was not a result of limiting reductase since cytochrome c reductase activities of liver and kidney microsomes were comparable (0.14 and 0.11 mmol cytochrome c per micromole P450). Nor was it a result of a lower affinity for the substrate since the Kms for ethoxyresorufin of liver and kidney microsomes were about the same (0.29 and 0.21 µM, respectively.)

	Discussion

Abstract Introduction Results Discussion References

Although TCDD induces two CYP1A enzymes in avian as well as in mammalian liver, the amino acid sequences of the chick enzymes do not permit either form to be classified as a 1A1 or a 1A2 ortholog (13). Nevertheless, CYP1A4 seems to exhibit more CYP1A1-like activities based on its selectivity for EROD and AHH while the other form, CYP1A5, seems to behave in some, but not all respects, more like CYP1A2 (11). Thus CYP1A5 shares an immunologic epitope with rat CYP1A2 but not 1A1 (11) and is more active in estradiol 2-hydroxylation than CYP1A4³. Here we show that purified CYP1A5 shares another characteristic with CYP1A2 in that it preferentially exhibits the CYP1A2-selective activity, UROX. Notably, the relative UROX activities exhibited by purified CYP1A5 and CYP1A4 shown here, 8:1, were the same as found for mouse CYP1A2 and 1A1 (4). Considering that CYP1A5 and CYP1A4 are present in approximately equal amounts in microsomes from TCDD-treated chick embryos (11), our findings lead to the conclusion that CYP1A5 is the main CYP enzyme responsible for the induced UROX expressed in chick embryo liver microsomes.

CYP1A5 does not appear to be a CYP1A2-like ortholog. Its amino acid sequence is more like CYP1A1 than 1A2 (13), and there are some respects in which CYP1A5 does not behave like CYP1A2. CYP1A5 is isolated in a low spin iron state² (9) and is insensitive to induction by isosafrole⁴ (11).

The finding that UROX and EROD were co-induced in the studies with both chick embryo microsomes and cultured cells (fig. 3) further distinguishes the avian CYP1A enzymes from mammalian CYP1A1 and 1A2. The mammalian enzymes exhibit different dose responses to various Ah receptor ligands in vivo (20-22). The coincident dose responses for EROD and UROX found here are in agreement with the findings for TCDD-induction of EROD and AA epoxygenation in liver of chick embryos (11) and suggest that the regulation of the two TCDD-induced chicken CYP1A enzymes differs in some respects from the regulation of mammalian CYP1A1 and 1A2.

Similarly, the finding of induced UROX activity in kidney from 3-methylcholanthrene-induced chickens is not characteristic of mammalian CYP1A2. CYP1A2 is scarcely expressed in rat kidney (2). The expression in chicken kidney of induced UROX is consistent with the observations that CYP1A5-mediated arachidonic acid epoxygenation is expressed in chick embryo kidney as well as liver (11). The expression of CYP1A5 in kidney and the evidence for relative differences in the amounts of CYP1A5 and CYP1A4 in liver and kidney provide further evidence supporting differences in the regulation of mammalian CYP1A1 and 1A2 and avian CYP1A4 and CYP1A5.

Both CYP1A5 and CYP1A4 were shown previously to be expressed and catalytically active in cultured chick embryo hepatocytes using arachidonic acid metabolism for CYP1A5 and EROD activity for CYP1A4 (19). The present results confirm the presence of both TCDD-induced enzymes in primary chick hepatocyte cultures, using EROD as an index of CYP1A4 and UROX as an index of CYP1A5. The results indicate that uroporphyrin accumulation in these cultures after treatment with Ah receptor ligands (15) was due to the ability of CYP1A5 to catalyze UROX and not to a single form of 3-methylcholanthrene-induced P450 which catalyzed both EROD and UROX, as was thought previously (2). The fact that chick embryo liver cultures express both avian CYP1A enzymes underscores the utility of the chick embryo culture system to study reactions involving both forms of P450 as well as the regulation of both forms. In contrast, primary rat hepatocytes, unlike chick embryo hepatocytes, do not accumulate uroporphyrin when treated with CYP1A inducers (7) because in the rat cultures CYP1A2 protein is not expresssed (23). Mouse hepatocyte cultures accumulate uroporphyrin, but only after several days of exposure to CYP1A inducers, coincident with increases in CYP1A2 (24, 25). TCB-induced accumulation is also found in a fish hepatoma line (26), indicating that those cells express a P450 that can catalyze UROX. Interestingly, Morrison et al. have cloned a single fish CYP1A that possesses some specific consensus sequences typical of mammalian CYP1A1 and 1A2 forms (27).

In summary, chicken CYP1A5 preferentially catalyzes UROX, an activity which has been previously shown to be a CYP1A2 activity in mammals. Thus, even though there are structural differences among mammalian and avian CYP1A enzymes, there is some conservation of the division of catalytic activities across different vertebrate classes. The challenge is to understand the basis for the division of function among CYP1A enzymes, and in particular, the selective relegation of UROX activity, an apparently toxic response, to one of two CYP1A enzymes.