Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

3-Methylcholanthrene (MC) is one of the most potent polycyclic aromatic hydrocarbon (PAH) carcinogens known (Harvey, 1982). Exposure of rodents to MC causes tumors of many organs, including the lung (Rasmussen et al., 1984) and mammary gland (Huggins et al., 1961). MC is an inert molecule by itself, but metabolism of MC by CYP enzymes leads to the formation of chemically reactive intermediates that are capable of binding covalently to DNA, a critical step in the initiation of carcinogenesis (Guengerich, 1988).

The CYP enzymes are a multigene family of hemoproteins of the microsomal mixed function oxidase system that is central to the activation and detoxication of carcinogens and other toxic chemicals and to the metabolism of a wide array of endogenous substances and xenobiotics, including drugs, pesticides, and organic pollutants (Guengerich, 1990). The CYP1A family comprises two proteins, CYP1A1 and 1A2, that play important roles in carcinogen activation (Guengerich, 1988). Recent studies have shown CYP1B1, a newly discovered enzyme, also to be active in PAH-mediated carcinogenesis (Bowes et al., 1996). The hepatic CYP1A1/1A2 enzymes are inducible by the hepatocarcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Whitlock, 1987), by PAHs such as MC (Thomas et al., 1983) and benzo[a]pyrene (BP) (Conney, 1986), and by cigarette smoke in humans (Kawajiri et al., 1991). Moreover, MC induces CYP1A1 in extrahepatic tissues such as lung (Nebert and Gelboin, 1969) and mammary tissues (Christou et al., 1995), which are target organs for MC-mediated carcinogenesis (Huggins et al., 1961; Rasmussen et al., 1984). A recent study has suggested that, in addition to its well established role of metabolism of polycyclic aromatic hydrocarbons and environmental pollutants, MC-inducible CYP1A1 may also participate in one or more critical life processes, such as cell division, migration, and apoptosis (Dey et al., 1999).

The mechanisms of induction of CYP1A1/1A2 by MC or TCDD have been investigated extensively (Safe, 1995; Denison and Phelan, 1998). The inducer, on entry into the cell, interacts with the Ah receptor (AHR), which is a cytosolic protein. The inducer-receptor complex is translocated into the nucleus, where it binds to the Ah receptor nuclear translocator (ARNT), and this complex in turn interacts with specific regulatory elements, termed Ah receptor elements (AhREs), 5' to the CYP1A1/1A2 and phase II enzyme [e.g., glutathione S-transferase-, NADP(H) quinone reductase] genes, resulting in enhanced expression of the CYP1A1, CYP1A2, and phase II enzymes encoded by the Ah locus (Safe, 1995; Denison and Phelan, 1998). In addition to the AhREs, several negative regulatory elements are located on the CYP1A1 promoter (Boucher et al., 1995).

The events occurring during the process of induction have been studied extensively, but relatively little is known regarding the return of drug-metabolizing enzymes to preinduction activities after cessation of xenobiotic exposure. In animals exposed to persistent chemicals such as TCDD (Gasiewicz et al., 1986), induction of CYP1A1 and phase II enzymes persists for several weeks after cessation of TCDD administration and is attributed to the long biological half-life of TCDD (Gasiewicz et al., 1986). Beebe et al. (1992) have demonstrated a prolonged induction of CYP1A1 enzyme in lungs of mice exposed to a single dose of Aroclor 1254, which correlates with retention of several polychlorinated biphenyls in tissues.

When induced by phenobarbital (Li et al., 1992) or -napthoflavone (Moorthy et al., 1994), the CYP enzymes return to normal within 5 days after withdrawal of treatment. In rats, a single dose of MC (20-25 mg/kg) induces aryl hydrocarbon hydroxylase (AHH) activity (Poland and Glover, 1974) and CYP1A1 mRNA level (Bresnick et al., 1981) after 24 h, followed by return of enzyme activity and mRNA levels within 5 to 7 days. However, Boobis et al. (1977) showed that a single dose of MC (80 mg/kg) increases total CYP contents and AHH activities in mice for up to 9 days. Masaki et al. (1984) reported increased total hepatic CYP contents in rats up to 8 days after multiple administrations of MC, and DePierre et al. (1982) showed induction of total hepatic CYP contents that persists for 2 to 3 weeks. However, mechanisms by which MC elicits sustained CYP1A1 induction have not been reported.

In this study, we investigated the mechanisms of sustained induction of CYP1A1 by MC. To this end, we tested the hypotheses that sustained induction of CYP1A enzymes in liver by MC is accompanied by up-regulation of CYP1A1/1A2 apoproteins and their mRNAs and that this phenomenon is mediated by mechanisms other than those due to long-term retention of the carcinogen in the body.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Chemicals

MC, calcium chloride, Tris, sucrose, NADP⁺, ethoxyresorufin, glucose 6-phosphate, and glucose-6-phosphate dehydrogenase were purchased from Sigma Chemical Co. (St. Louis, MO). Methoxyresorufin was purchased from Molecular Probes (Eugene, OR). Ring-labeled [³H]MC (radiochemical purity >99.9%) was obtained from ChemSyn Laboratories (Lawrence, KS). Polyvinylidene difluoride membranes and buffer components for electrophoresis and Western blotting were obtained from Bio-Rad Laboratories (Hercules, CA). The primary monoclonal antibody to CYP1A1 was a gift from Dr. P. E. Thomas (Rutgers University, New Brunswick, NJ). Goat anti-mouse IgG conjugated with horseradish peroxidase (HRP) was from Bio-Rad Laboratories. CYP1A1 cDNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA were gifts from Dr. Frank Gonzalez of the National Cancer Institute (Bethesda, MD) and Dr. T. Tamura of our department, respectively.

Animals

Two-month-old female Sprague-Dawley rats (180-200 g) were obtained from Harlan Sprague-Dawley (Houston, TX). The animals were acclimatized for 7 days before the start of the experiment and randomized into three groups of 6 to 10 animals each. Tap water and food (Purina Rodent Lab Chow 5001; Purina Mills, Richmond, IN) were available ad libitum. A 12-h light/dark cycle was maintained.

Animal Treatment

Female Sprague-Dawley rats were treated with MC (93 µmol/kg) in corn oil (CO) (2 ml/kg), or were given equal volumes of CO i.p., once daily for 4 days. At selected time points, animals were sacrificed, and microsomes were isolated immediately from portions of liver, lung, and mammary tissues, and stored at 80°C until analysis. The remainder of the livers were stored at 80°C for later isolation of RNA. Some CO- or MC-treated animal livers were used for isolation of nuclei. For dose-response studies, animals were administered [³H]MC (0-100 µmol/kg, 100 µCi/kg) and sacrificed 24 h after dosing. The livers were analyzed for CYP1A1/1A2 induction and levels of parent MC.

Preparation of Microsomes

The livers were excised, weighed, and homogenized in 10 mM Tris-HCl, pH 7.4, containing 0.25 M sucrose; microsomes were prepared by the calcium chloride precipitation method (Cinti et al., 1972). Microsomes from lungs and mammary glands were prepared by differential centrifugation (Matsubara et al., 1974). Lungs were perfused with cold saline before excision and subsequent preparation of microsomes. Protein concentrations were estimated by the Bradford dye-binding method (Bradford, 1976).

Isolation of Nuclei

Nuclei and nuclear protein extracts from livers of CO- or MC-treated animals were prepared according to the procedure of Okino et al. (1992). The nuclear protein extracts were stored at 80°C until use.

Enzyme Assays

Total CYP contents and EROD and methoxyresorufin O-demethylase (MROD) activities in microsomes were assayed essentially as described previously (Moorthy et al., 1993, 2000).

Electrophoresis and Western Blotting

Liver microsomes (30 µg of protein) were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) in 7.5% acrylamide gels, followed by Western blotting, as reported in a recent paper from our laboratory (Moorthy et al., 2000). Detection of CYP1A1/1A2 proteins on the Western blots was accomplished by the use of a monoclonal antibody to CYP1A1 that cross-reacts with CYP1A2 as the primary antibody and goat anti-mouse IgG conjugated with HRP as the secondary antibody (Moorthy et al., 2000). The blots were developed with the HRP color reagent 4-chloro-1-naphthol (Moorthy et al., 2000).

Northern Hybridization

Total liver RNA was isolated using a modification of the procedure of Chomczynski and Sacchi (1987). The RNA (20 µg/sample) was loaded onto a 1% agarose/formaldehyde denaturing gel, separated by electrophoresis, and transferred to nitrocellulose filters. The filters containing separated RNA were prehybridized by exposure to 20 mM Na₂HPO₄, pH 7.6, 10× Denhardt's reagent, 100 µg/ml heat-denatured salmon sperm DNA, and 7% SDS for approximately 3 h at 55°C. After prehybridization, the solutions were discarded, and nitrocellulose filters were hybridized at 55°C for 16 h by using the same solution used for prehybridization and adding 10% dextran and the ³²P-labeled CYP1A1 cDNA probe (20 × 10⁶ cpm) that was prepared by the random prime technique (Hamburg et al., 1994). The hybridized filters were washed three times for 20 min with 3× sodium chloride sodium citrate (SSC) (1× SSC, 0.15 M sodium chloride, 0.015 M sodium citrate), and 1% SDS at 68°C to decrease nonspecific binding of radioactive transcripts to the filter. After these washes, the membranes were exposed to autoradiography. Relative levels of CYP1A1/1A2 mRNAs were quantitated by phosphorimager analysis. GAPDH cDNA probe was used as an RNA transfer and loading control. For this, the nitrocellulose membranes were stripped and hybridized with GAPDH cDNA probe, followed by exposure to autoradiography.

Uptake, Distribution, and Excretion of [³H]MC

Uptake of [³H]MC into Liver. A single dose of [³H]MC (372 µmol/kg) (specific activity, 0.86 mCi/mmol) was administered i.p. to female Sprague-Dawley rats. The animals were sacrificed at selected time points, from 4 to 24 h after treatment. The livers were dissected and homogenized, and aliquots corresponding to 100 mg of liver were solubilized in Soluene 350, mixed with 15 ml of Hionic Fluor, and counted for radioactivity using a Packard liquid scintillation counter. Quenching corrections were calculated from quench curves generated by the machine. Soluene 350, Hionic Fluor, and the scintillation counter were purchased from Packard Instrument Company (Meriden, CT).

Disappearance of [³H]MC-Derived Radioactivity from Liver and i.p. Fat. Twelve animals were administered [³H]MC (93 µmol/kg; specific activity, 0.86 mCi/mmol) i.p., once daily for 4 days. Three animals from each group were sacrificed at 1, 8, or 15 days after the last treatment. Hepatic levels of [³H]MC were determined as described above. For determination of levels of [³H]MC in i.p. fat, fatty tissues were removed from the mesenchymal, epididymal, and mesenteric tissues and homogenized in 10 mM PBS, pH 7.4; 100-mg portions were processed for measurements of radioactivity.

Estimation of Intrahepatic Concentrations of Parent [³H]MC. The liver tissues of [³H]MC-exposed animals were homogenized in 10 mM Tris-HCl buffer, pH 7.4, and extracted three times with 2 volumes of ethyl acetate. The ethyl acetate fractions were combined, concentrated, and chromatographed on Eastman Kodak silica gel thin-layer plates with fluorescent indicator. The solvent system was hexane:ethyl acetate, 9:1. A small amount of unlabeled MC (1-2 µg) was spotted alongside the biological samples containing [³H]MC and its metabolites to locate on the chromatogram, under UV light, the parent [³H]MC. The R_f of MC was 0.67. The chromatogram was cut into several rectangular zones, and the amount of radioactivity in each zone was measured in a scintillation counter. The radioactivity in the zone corresponding to R_f of 0.67, which represented the parent [³H]MC, was used to estimate the amount of parent [³H]MC in liver.

Excretion of [³H]MC from Rats. Animals were housed in metabolic cages for collection of feces and urine. The animals were given [³H]MC (93 µmol/kg; specific activity, 0.86 mCi/mmol) once daily for 4 days. Urine and feces were collected daily for up to 15 days after cessation of MC treatment. The feces were homogenized in water, aliquots were heated in Soluene 350 for 4 to 6 h at 68°C, and evels of radioactivity were determined by liquid scintillation counting Radioactivity in urine was measured by mixing a 100-µl aliquot with 15 ml of Hionic Fluor, followed by counting of radioactivity.

Concentration of [³H]MC Remaining in Body Organs and Carcasses. Concentrations of [³H]MC and its metabolites remaining 15 days after cessation of MC treatment were estimated by dissection of all major organs, followed by processing of the tissues for radioactivity measurements, as described for liver. The remaining carcasses were dehydrated by heating in a vacuum oven and ground using a mortar and pestle; the resulting powder was analyzed for radioactivity. The results are expressed as percentage of dose, to reflect whole organ distribution, and per gram of tissue, to reflect the relative concentrations of the chemical.

Electrophoretic Mobility Shift Assays (EMSA)

EMSAs were performed as described by Okino et al. (1992). Briefly, the nuclear proteins (15 µg) were preincubated with poly(dI-dC) (2.5 µg) on ice for 10 min, followed by incubation at room temperature for 20 min with 75,000 cpm of -³²P-labeled double-stranded oligonucleotides that contain the AhREs (Pendurthi et al., 1993) in the presence of EMSA buffer (25 mM HEPES, 1 mM EDTA, 1 mM DTT, 10% glycerol, 500 mM KCl, pH 7.5). In some experiments, the nuclear extracts were incubated with a 50-fold excess of cold AhRE-specific oligonucleotide, before the addition of the -³²P-labeled probe. The labeled samples were separated by nondenaturing PAGE (4% gels) at 200 V for 2 h. The gel was dried, and radioactivity was located by autoradiography using Kodak X-ray film.

Statistical Analysis

Data obtained from individual animals are expressed as means ± S.E. Statistical analysis was performed by ANOVA. Two-way ANOVA was performed to determine the effect of MC on EROD or MROD activities at selected time points, with MC and time as the two dependent variables. Statistical significance between control and treated groups for each time point was assessed by modified t tests, with P < .05 being considered significant. One-way ANOVA was performed to determine the effect of MC on CYP1A1 or CYP1A2 mRNA levels. Statistical significance between different groups was assessed by Tukey's post hoc test, with P < .05 being considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Resorufin Ether O-Dealkylase Activities

On the 1st day after four daily doses of MC, treated rats showed hepatic EROD activities that were 48 times the activities observed in animals treated with vehicle not containing MC (Fig. 1). The EROD activities remained higher than vehicle-treated controls through day 28 (Fig. 1). Two-way ANOVA revealed a significant effect on EROD activities of MC treatment and time and a significant interaction between the two factors. MC also induced MROD activities. At the 1-day time point, MC induced MROD activities by 12-fold over controls (Fig. 1), and this induction persisted for up to 15 days (Fig. 1). Similar to EROD data, ANOVA showed a significant effect on MROD activities of treatment with MC and time, as well as a significant interaction between the two factors. The sustained elevation of EROD and MROD activities was noticed also in animals given a single dose (372 µmol/kg) of MC (data not shown).

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Effects of treatment with MC on hepatic EROD and MROD activities in rats. Female Sprague-Dawley rats (200 g) were treated i.p. with MC (93 µmol/kg) dissolved in 2 ml/kg CO or vehicle only as control (C) once daily for 4 days and the animals were sacrificed 1, 8, 15, 28, or 45 days after last treatment. EROD and MROD activities were determined in liver microsomes as described in Materials and Methods. Each value represents mean ± S.E. of activities from four individual animal livers. Data were analyzed by two-way ANOVA; control and MC-treated samples were compared by modified t tests for each time point. **, ***, and ****, different from vehicle-treated animals (C) at P < .01, P < .001 and P < .0001, respectively.

CYP1A1/1A2 Apoproteins

As seen in Fig. 2, induction of both CYP1A1 and 1A2 proteins was observable by Western analysis for up to 45 days in MC-treated rats. CYP1A2, but not CYP1A1, was readily detectable in control liver. Densitometric analysis (data not shown) indicated that the CYP1A1 protein levels were 95 and 90% of the 1-day value at 8 and 15 days, respectively. By days 28 and 45, the protein levels declined to 30 and 15% of the 1-day value, respectively. CYP1A2 protein induction was also sustained at extents similar to those observed for CYP1A1 (data not shown).

View larger version (89K): [in this window] [in a new window]   Fig. 2.   Western analysis of CYP1A1 and CYP1A2 apoproteins. Liver microsomes (20 µg) from control and MC-treated rats (see Fig. 1 for details on animal treatments) were used to analyze CYP1A1 and 1A2 apoprotein contents by Western analysis. Lane 1, molecular mass markers; lane 2, microsomes from control animals; lanes 3 through 7, microsomes from MC-treated animals at 1, 8, 15, 28 and 45 days, respectively. The slow- and fast-moving bands seen in the 50-kDa region on lanes 3 to 7 correspond to CYP1A1 and 1A2 proteins, respectively.

CYP1A1/1A2 mRNA Levels

To determine whether the sustained induction of CYP1A1/1A2 enzyme activities and apoprotein contents was accompanied by corresponding increases in mRNA levels, we performed Northern hybridization analysis on total RNA from livers isolated from control and MC-exposed animals with cDNA probes for CYP1A1, which cross-hybridizes with CYP1A2 mRNA. As shown in Fig. 3, at the 1-day time point, MC samples displayed a significant induction of the CYP1A1 (23S) and CYP1A2 (18S) mRNAs, respectively. Notably, a clear hybridization band was obtained at 18S with normal liver RNA. Quantitation of mRNA levels by a phosphorimaging technique revealed that MC induced CYP1A1 and 1A2 mRNAs by 25.4- and 4.4-fold, respectively, over the corresponding controls (Fig. 4). At the 15-day time point, MC-treated animals continued to display elevated mRNA levels, with CYP1A1 and 1A2 levels being 16- and 3-fold, respectively, over controls.

View larger version (53K): [in this window] [in a new window]   Fig. 3.   Effect of MC on hepatic CYP1A1 and CYP1A2 mRNA levels. Animals were treated with MC or CO vehicle alone as control (C) as described in Fig. 1, and total RNA was isolated from livers of rats 1 or 15 days after the last treatment. The RNA was electrophoresed, transferred, and probed with random prime 32P-labeled CYP1A1 cDNA. GAPDH cDNA was used as an RNA transfer and loading control. Film exposure was for 24 h at 80°C.

View larger version (42K): [in this window] [in a new window]   Fig. 4.   Quantitative analysis of CYP1A1 and CYP1A2 mRNA. The CYP1A1 and CYP1A2 mRNA species shown in the Northern blot (Fig. 3) were quantitated using a phosphorimager, and mRNA levels were calculated. Each bar represents means ± S.E. of pixel densities of the bands corresponding to CYP1A1 and 1A2 mRNAs in control (C) and MC-treated animals from three individual animal livers. CYP1A1 and CYP1A2 data were separately analyzed by one-way ANOVA, followed by Tukey's post hoc test. ***, different from control and each of the MC-treated groups at P < .001.

Persistent Induction of CYP1A1 in Extrahepatic Tissues

The effect of MC on EROD activities in lung at selected time points is shown in Fig. 5. Comparison of EROD activities between MC-exposed and control animals revealed a 55-fold induction of EROD by MC at 1 day (Fig. 5). On day 15, the MC samples continued to display induction of EROD activities, being 56-fold greater than the corresponding controls. In mammary tissues, comparison of EROD activities between MC-treated and control animals indicated 7-fold greater activities in MC samples versus controls at 1 day (Fig. 6). At the 15-day time point, the EROD activities in the animals exposed to MC were 3.8-fold higher than controls.

View larger version (45K): [in this window] [in a new window]   Fig. 5.   EROD activities in rat lung microsomes. Rats were exposed to vehicle or MC as described in the legend to Fig. 1, and the animals were sacrificed at 1 or 15 days after MC withdrawal. EROD activities in control (C) or MC-exposed lung microsomes were assayed as described under Materials and Methods. Values represent means ± S.E. of activities from three individual samples, each sample having been generated by pooling tissues from three individual animals. Statistical analysis was performed by two-way ANOVA, and values from control and MC-treated animals were compared by modified t test. **** denotes statistically significant differences between control and MC-treated samples at P < .0001.

View larger version (41K): [in this window] [in a new window]   Fig. 6.   EROD activities in rat mammary microsomes. Rats were exposed to vehicle CO or MC as described in the legend to Fig. 2, and the animals were sacrificed at 1 and 15 days after MC withdrawal. EROD activities were assayed in the control (C) and MC-treated mammary microsomes. Values represent means ± S.E. of activities from three individual samples, each sample having been generated by pooling tissues from three individual animals. Statistical analysis was performed by two-way ANOVA. ** and *** denote statistically significant differences between control and MC-treated samples at P < .01 and P < .001, respectively.

Uptake, Distribution, and Excretion of [³H]MC

Uptake of [³H]MC by Liver. The uptake of [³H]MC by the liver after i.p. administration of a single dose of [³H]MC is presented in Fig. 7. After a small lag, a rapid and linear increase in the concentration of MC in liver was observed. By 24 h, the level of MC and its metabolites corresponded to 1.5% of the administered MC dose.

View larger version (11K): [in this window] [in a new window]   Fig. 7.   Uptake of [3H]MC by liver in vivo. Female Sprague-Dawley rats were administered a single dose of [3H]MC (372 µmol/kg; specific activity, 0.86 mCi/mmol) i.p. in CO (2 ml/kg). At the times indicated, animals were sacrificed and 1-g portions of livers were homogenized in PBS (10 mM), pH 7.4. Aliquots corresponding to 100 mg were processed for radioactivity measurements. Values represent means ± S.E. of duplicate assays from three individual animals.

Disappearance of [³H]MC from Liver. To determine the rate of disappearance of [³H]MC from liver under conditions used to study sustained induction of CYP1A1/1A2 by MC, we studied the profile of total radioactivity in liver after four i.p. doses of [³H]MC. One day after the last treatment, only 0.94% of radioactivity was recovered in liver (Fig. 8), in contrast with the 1.5% noticed after a single dose of MC (372 µmol/kg) (Fig. 7), suggesting that MC stimulated its own metabolism. At 8 days, the radioactivity fell to 0.3% of the total dose, and by 15 days, the total radioactivity represented 0.09% of the administered dose (Fig. 8). The measured radioactivity represents free MC, its metabolites, and metabolites bound to cellular macromolecules. To estimate the intrahepatic concentration of parent MC at the 15-day time point, the liver tissues were extracted with ethyl acetate, and the ethyl acetate extract was chromatographed by thin-layer chromatography. As shown in Table 1, the intrahepatic concentration of parent MC, 15 days after cessation of MC treatment, was 270 pmol/g.

View larger version (13K): [in this window] [in a new window]   Fig. 8.   Time course of disappearance of [3H]MC from the liver. Rats were exposed to [3H]MC (93 µmol/kg; specific activity, 0.86 mCi/mmol) in CO (2 ml/kg) once daily for 4 days, and animals were sacrificed at 1, 8, or 15 days after drug withdrawal. Liver tissues were processed for radioactivity measurements. Radioactivity values are expressed as percentages of total MC dose. The values represent means ± S.E. of duplicate assays from three individual animals.

Disappearance of Radioactivity from i.p. Fat. As shown in Fig. 9, the radioactivity from i.p. fat also disappeared rapidly, as evidenced by the concentration of MC and its metabolites dropping from 1.03% of the administered dose at the 1-day time point to 0.11% at the 15-day time point (Fig. 9).

View larger version (10K): [in this window] [in a new window]   Fig. 9.   Time course of disappearance of [3H]MC from i.p. fat. The fatty tissues from the i.p. cavities of [3H]MC-exposed rats were blotted, weighed, homogenized in PBS (10 mM, pH 7.4), and processed for radioactivity measurements. Radioactivity values are expressed as percentages of total MC dose. The values represent mean ± S.E. of duplicate assays from three individual animals.

Excretion of [³H]MC from Rats. Although the above-mentioned data suggested extensive metabolism of MC in liver, the possibility of sequestration of MC into different storage sites (e.g., fatty tissues) was not totally excluded. Therefore, we performed experiments to determine the excretion rate of MC from rats. Animals housed in metabolic cages were administered [³H]MC as described under Materials and Methods. Urine and feces were collected daily for up to 15 days after cessation of MC treatment. The total recovery was 93%. Of the 93 µmol of MC administered, approximately 50 µmol of MC and its derivatives were recovered in feces and urine during the 4-day treatment period. This represented 54% of the given dose (data not shown). On the 1st day after MC withdrawal, 10 µmol of MC and its metabolites were present in the feces. By day 2, more than 68% of the administered dose had been excreted (Fig. 10). By day 15, more than 92.3% of [³H]MC had been excreted, with feces being the major route of excretion of radioactivity (Fig. 10).

View larger version (16K): [in this window] [in a new window]   Fig. 10.   Excretion of [3H]MC from rats. Rats were dosed with [3H]MC as described in Fig. 8 once daily for 4 days. Urine and feces were collected daily for up to 15 days after MC withdrawal, and the samples were processed for measurements of radioactivity. Data in the figure depict radioactivity excreted from days 1 to 15 after MC withdrawal. Data represent mean + S.E. from two individual animals.

Dose-Response Studies. As shown in Table 3, a dose-dependent induction of EROD and MROD activities was noticed 24 h after a single dose of MC. No significant induction of total CYP contents or EROD/MROD activities was seen at doses of MC ranging from 0 to 2 µmol/kg. The mean intrahepatic concentration of parent MC, 24 h after administration of MC (2 µmol/kg), was 271 pmol/g (Table 3). Administration of MC (5 µmol/kg), which produced an intrahepatic MC concentration of 525 pmol/g after 24 h, led to 1.5-, 8.9-, and 3.1-fold induction of hepatic CYP contents, EROD and MROD activities, respectively (Table 3). Treatment of animals with doses of MC greater than 5 µmol/kg led to greater increases of total CYP contents and EROD and MROD activities, reaching the maximum level of induction at a dose of 50 µmol/kg, which produced an intrahepatic MC concentration of 1850 pmol/g.

Nuclear MC-AhR Complexes In Vivo

Specific binding of the AHR to AhREs can be determined by EMSA (Okino et al. 1992). To determine whether sustained induction of hepatic CYP1A1/1A2 by MC is dependent on the interaction of ligand-activated AhR in the nucleus with AhREs on the CYP1A1 promoter, we performed EMSAs using the nuclear protein extracts from CO- (Fig. 11, lane 1) and MC- (Fig. 11, lanes 2-4) treated animals. As shown in lane 2, a nuclear protein that binds to the AHREs was identified in livers of rats 24 h after termination of treatment with MC (93 µmol/kg), which was administered i.p., once daily for 4 days. The shifted band was competed off in the presence of 50-fold excess of AhRE-specific oligonucleotide (lane 4). This protein was not observed in rats treated with CO (lane 1) or in animals 15 days after termination MC treatment (lane 3).

View larger version (55K): [in this window] [in a new window]   Fig. 11.   Representative EMSA of nuclear extracts from CO- and MC-treated rats. Female Sprague-Dawley rats (200 g) were treated i.p. with MC (93 µmol/kg) (lanes 2-4) or CO (2 ml/kg) (lane 1) once daily for 4 days, and the animals were sacrificed at 1 (lane 2) or 15 days (lane 3) after last treatment. Nuclear protein extracts from livers of these animals were subjected to EMSA, as described under Materials and Methods. The labeled nuclear proteins were separated by PAGE, and the gels were dried. The gels were exposed to autoradiography at 80°C for 16 h. Arrow indicates interaction with the AhREs of an MC-specific nuclear protein that appears to be the MC-AHR complex (lane 2), which is competed off in the presence of a 50-fold excess of cold AhRE-specific oligonucleotide (lane 4).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In this study, we investigated the mechanisms of long-term induction of CYP1A1/1A2 by MC in rats. The dramatic increase in EROD and MROD activities at the 1-day time point in livers of animals exposed to MC indicated induction of CYP1A1 and 1A2 enzymes, on the basis of evidence that EROD and MROD activities are relatively specific for CYP1A1 and 1A2, respectively (Moorthy et al., 2000). Our data (Fig. 1) showing statistically significant increases in EROD and MROD activities for several weeks after cessation of MC treatment suggested that MC elicited a prolonged induction of CYP1A1/1A2 enzymes (Fig. 1).

The fact that sustained induction of the enzyme activities was paralleled by persistent induction of the apoproteins for CYP1A1 and 1A2 (Fig. 2) supports the idea that the phenomenon of persistent induction of CYP1A1/1A2 activities was a result of long-term induction of their corresponding apoproteins. Because our Northern hybridization experiments showed sustained up-regulation of CYP1A1/1A2 mRNA levels after treatment with MC (Figs. 3 and 4), it is unlikely that MC caused persistent induction of CYP1A1/1A2 primarily by inhibiting the degradation of the corresponding apoproteins, although this mechanism may have, in part, contributed to the sustained CYP1A1 induction by MC. The sustained increase in the levels of mRNAs for CYP1A1 and 1A2 by MC was presumably due to enhanced rates of transcription of the corresponding genes. The possible contribution of message stabilization to the persistent augmentation of CYP1A1/1A2 mRNA levels has not been excluded, and it is possible that post-transcriptional mechanisms may have also contributed to the sustained induction.

The observation that MC elicited sustained induction of EROD (CYP1A1) activities in extrahepatic tissues such as the lungs (Fig. 5) and mammary glands (Fig. 6) suggested that this phenomenon is of relevance to carcinogenesis because lungs and mammary glands are target organs for the carcinogenesis mediated by MC and other PAHs (Huggins et al., 1961; Rasmussen et al., 1984). The findings of Beebe et al. (1992) demonstrating a correlation between persistent induction of CYP1A1 enzyme in the lungs of mice exposed to the tumor promoter Aroclor 1254 indicate the significance of sustained CYP1A1 induction to tumorigenesis. We had earlier reported that MC did not elicit sustained induction in kidney, a nontarget organ for PAH-mediated carcinogenesis (Li et al., 1992), suggesting that the prolonged induction in lung and mammary gland may have implications for carcinogenesis.

We reported earlier (Moorthy et al., 1994) that the environmentally prevalent compound dibenz[ac]anthracene (DBA) also elicits a long-term induction of CYP1A1/1A2 in rat liver. On the other hand, BP and naphthacene do not elicit a prolonged induction (Moorthy et al., 1994). Because both MC and DBA possess a benz[a]anthracene bay region, we hypothesized that a benz[a]anthracene bay region may be an important structural requirement in mediating the sustained response. If PAHs can elicit sustained induction of CYP1A1 in lungs and/or mammary tissues of humans, it is possible that this phenomenon may contribute to human cancers of the lung and/or breast.

Our data pertaining to the uptake, distribution, and elimination of [³H]MC in vivo (Figs. 7-10, Tables 1 and 2) revealed rapid metabolism and clearance of the carcinogen from the rat. As shown in Fig. 8, only 0.94% of the administered dose was recovered in liver 1 day after termination of MC treatment, suggesting that MC was metabolized and eliminated quickly from liver. The observation that, of a total recovery of 93%, greater than 92.3% of radioactivity was recovered in feces and urine (Fig. 10) suggests that MC was almost entirely excreted from the body and was not sequestered into different storage sites of the body. The fact that the bulk of the excreted radioactivity (~97%) was associated with the feces (Fig. 10) was consistent with extensive hepatic metabolism of MC, followed by biliary excretion and elimination via the feces (Daniel et al., 1967). The observation that little radioactivity (~3%) was excreted in the urine over the 15-day time period (Fig. 10) excluded the possibility that in vivo tritium exchange of [³H]MC with water may have resulted in underestimation of the levels of MC that persisted in the body. Overall, the results were consistent with the experiments of Bresnick et al. (1967) who recovered only 0.5% of the dose in liver 14 h after a single dose of MC. Furthermore, Levine and Singer (1972) have shown that i.v. administration of MC leads to swift metabolism and elimination of the carcinogen. Other PAHs such as BP and 7,12-dimethyl benz[a]anthracene are also eliminated rapidly from rats (Daniel et al., 1967).

The residual concentrations of MC and its derivatives in our studies were approximately 0.09 µmol in whole liver and 0.45 µmol in the whole body at the 15-day time point (Fig. 8). The concentration of parent MC in liver at this time point was 270 pmol/g (Table 1). The fact that administration of a single dose of MC (2 µmol/kg), which produced an intrahepatic concentration of 271 pmol/g after 24 h, did not cause a significant induction of CYP1A1/1A2 (Table 3) strongly suggests that the sustained induction of CYP1A1/1A2 by MC is mediated by mechanisms other than persistence in the liver of the parent carcinogen. Furthermore, the observation that an intrahepatic MC concentration of 1850 pmol/g, produced 24 h after the administration of a single i.p. dose of MC (50 µmol/kg), was required to elicit a 32-fold induction of hepatic EROD activities (Table 3) lends credence to the hypothesis that the 37-fold elevation of EROD activities noticed 15 days after cessation of MC treatment (Fig. 1), when the intrahepatic MC concentration was 270 pmol/g, was not due to the persistence of the parent carcinogen in liver. This was in contrast to chemicals such as TCDD and polychlorinated biphenyls, which elicit prolonged induction CYP enzymes due to retention of the chemicals in body tissues. Thus, the results of this study were consistent with the working hypothesis that prolonged CYP1A1/1A2 induction by MC was caused by mechanisms other than those due to persistent retention of carcinogen in liver.

The band shift in MC-treated animals at 24 h but not in control animals (Fig. 11), as detected by EMSA, most probably represented interaction of the MC-AHR-ARNT complex with AhREs of the CYP1A1 promoter. The fact that the band shift was no longer observed in animals 15 days after MC withdrawal suggests that AHR-independent mechanisms significantly contributed to the long-term induction of CYP1A1/1A2 by MC. We have not excluded the possibility that, at later time points, a metabolite of MC or an endogenous ligand activated the AHR, which in turn interacted with some regulatory elements on the CYP1A1 promoter that were not present in the oligo probes that we used in our EMSAs.

The mechanisms of sustained induction of CYP1A enzymes by MC are not understood. It is possible that certain types of chemical stress response(s) associated with PAH treatment may have contributed to the sustained induction after MC exposure. As regards the lack of CYP1A induction after administration of a low dose of MC (2 µmol/kg), it is conceivable that this phenomenon was, in part, due to an inflammatory response. Because it is known that cytokine release is associated with PAH treatment, it is likely that the cytokine-mediated down-regulation of CYP enzymes contributed to the lack of CYP1A induction.

Regardless of the mechanism of persistent induction of CYP1A1/1A2 by MC, the phenomenon of sustained induction by MC may have significant implications for carcinogenesis by PAHs. These implications are: 1) because CYP1A1/1A2 are involved in the metabolic activation of PAHs to DNA-binding metabolites, exposure to a persistent inducer may lead to increased or decreased genotoxic risk on subsequent exposure to other genotoxic carcinogen(s). 2) Persistent induction may enhance metabolism of PAHs, leading to generation through redox cycling of increasing amounts of reactive oxygen species which may cause oxidative DNA damage (Ichinose et al., 1997). 3) As CYP1A enzymes are involved in estrogen metabolism (Safe, 1995), long-term induction of CYP may enhance the production of estrogenic metabolites, some of which are tumor promoters.

In conclusion, the results of this study strongly suggest that MC elicits persistent induction of CYP1A1/1A2 by mechanisms other than persistence of the parent carcinogen and probably involves AHR-independent mechanisms. A recent study (Dey et al., 1999) suggesting an important role for CYP1A1 in regulating critical life processes, in addition to its role in carcinogenesis by PAHs, calls for further in-depth investigations to elucidate the molecular mechanisms of persistence of MC-inducible CYP1A1/1A2, a phenomenon which potentially has important implications for carcinogenesis as well as other important biological processes.