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Studies of the expression of the cytochrome P450IA, P450IIB, and P450IIC gene family in extrahepatic and hepatic tissues.

We have studied the expression of three P-450 gene subfamilies in hepatic and extrahepatic tissues using the sensitive RNAse A protection assay. Members of the P450IA subfamily, which encodes the major methylcholanthrene-inducible cytochromes P-450, were found to be not expressed in extrahepatic tissues of untreated animals, raising the question whether these P-450 play a role in the metabolism of carcinogens in unexposed individuals. In contrast, members of the P450IIB family, some of which encode the major phenobarbital-inducible cytochromes P-450, were found to be expressed in some extrahepatic tissues of untreated rats and here most notably in the lung and in sebaceous glands. Members of the P450IIC family, which encode some constitutively expressed cytochromes P-450, were found to be expressed exclusively in the liver. ImagesFIGURE 1.FIGURE 2.FIGURE 3.


Introduction
Cytochromes P-450 are a large family of monooxygenases that play an important role in the metabolism of a wide variety of endogenous and exogenous compounds including steroids and polycyclic hydrocarbons (1). Recently several P-450 cDNAs and genomic clones have been isolated and sequenced, making it possible to order the various cytochrome P-450 genes in families and subfamilies on the basis of sequence comparisons (2,3).
Cytochromes P-450 have been mainly studied in the liver, which is certainly not the target organ for most chemical carcinogens. This approach has been chosen because the liver consists mainly of a single cell type, the hepatocyte, and because this organ contains a considerable amount of several cytochromes P450, some of which can be induced by xenobiotics. Only very few studies have been concerned with cytochromes P450 in extrahepatic tissues (4)(5)(6). In these studies antibodies against purified cytochrome P450 isozymes have been used.
Only recently specific oligomer probes for cytochrome P-450 genes have been used to study their expression in several tissues (7). However, experiments involving oligonucleotides will only show whether a given probe is able to recognize an mRNA, but will usually not reveal the extent of recognition. Thus, these methods are not useful for the identification of a previously unknown member of a given cytochrome P-450 gene family. On the other hand, Myers and Maniatis (8) have developed a method based on the RNAse A protection assay which will allow the detection of single point mutations in large stretches of the 3-globine gene. In this assay a radiolabeled anti-sense RNA probe, which is generated in vitro, is hybridized to RNA isolated from a tissue. The resulting duplex molecules are treated with RNAse A, which is known to cut into imperfectly matched duplex molecules.
With this method we have studied the expression of the P450IA family, which encodes the major methylcholanthrene-inducible cytochromes P-450; the expression of the P450IIB family, which encodes the major phenobarbital-inducible cytochromes P-450; and the expression of the P450IIC family, which encodes some cytochromes P-450 that are constitutively expressed in several tissues.

Results
Tissue-Specific Expression of the P4501A Family The P450IA1 (1957-2620) transcript (numbers in parentheses indicate the start and the end ofthe transcript numbered from the initiation codon) was used for the RNAse A protection assay with RNA isolated (9) from several tissues (Fig. 1). The in vitro transcribed antisense RNA (Fig. 1, lane 14) consisted mainly of a full length transcript. RNA isolated from the liver of Aroclor 1254-treated animals but not that from untreated animals protected a large amount of the probe (Fig. 1, lanes 1 and 2 vs. lane 3). RNA from the lung and the kidney of Aroclor-treated animals protected considerably less RNA probe than did hepatic RNA from treated animals. Only a weak signal was obtained with RNA from the intestinal mucosa of Aroclor-treated animals (Fig. 1, lane 10). No signal was obtained with RNA isolated from the kidney and the intestinal mucosa of untreated animals nor with the RNA isolated from the testis of untreated and treated rats nor from the brain of male and female rats.

Tissue-Specific Expression of the P450IlB Family
The P450IIB1 (133-499) anti-sense RNA was used in the nuclease Si and RNAse A analysis of RNA isolated from several tissues. Nuclease Si protection assay of mRNA from untreated animals and of Aroclor 1254treated animals (Fig. 2) yielded a protected fragment which had the same size as the fragment generated in the protection assay of the in vitro transcribed unlabeled P450IIB1 (133-499) sense transcript (Fig. 2). As expected, the amount of this fragment increased follow- ing Aroclor treatment. The RNAse A protection assay of mRNA isolated from Aroclor-treated animals yielded an intense signal for a fragment that had a slightly lower size than the fragment seen in the nuclease Si protection assay (Fig. 2). A very small amount of this fragment was also seen in the RNAse protection assay of mRNA from control animals; however, this RNA yielded several additional fragments that were hardly seen in the analysis of mRNA isolated from Aroclor-treated animals (Fig. 2). This result clearly shows that the liver of untreated animals contained a P450IIB1-related mRNA that was suppressed by Aroclor treatment. mRNA isolated from the lung and the intestine of treated animals (Fig. 2) yielded the same RNAse Aprotected fragment as the in vitro transcribed P450IIB1 (133-499) sense transcript. mRNA isolated from the testis, the brain, and the kidney did not give any distinct RNAse Aor nuclease Si-protected fragment. However, RNA isolated from the preputial gland yielded a large amount of several partially RNAse A-protected fragments that were distinct from the fragments obtained in the analysis of RNA isolated from control animals.

Tissue-Specific Expression of the P45011C Family
Two probes were used to study the expression of the genes of the P450IlC family. One probe was complementary to the P450IIC6 cDNA, the other probe was complementary to the P450IIC7 cDNA. With the P450IIC7 (613-763) probe, a signal was only obtained with hepatic RNA from male and female rats, independent of whether or not they had been treated with Aroclor 1254 (data not shown).
With the P450IIC6 (556-763) probe, two protected fragments were only obtained with hepatic RNA from untreated animals (Fig. 3). These protected fragments were distinctly smaller than the fragment protected by the in vitro transcribed P450IIC6 (556-763), indicating that the probe hybridized to a hepatic RNA which was not completely complementary to it.

Discussion
The P450IA family consists of two members, termed P45OIA1 and P450IA2. These two genes appear to code for two proteins that were named cytochrome P-450c and d (10). Early immunological data of Guengerich and Mason (4) showed that these proteins occur in the liver of untreated animals and are strongly induced by methylcholanthrene. In addition, these enzymes have been found in several extrahepatic tissues. However, cytochrome P45OIA1 and IA2 were not analyzed differentially. In a recent study (5), the amount of cytochrome P45OIA1 protein was determined selectively in several tissues. In contrast to our study (Fig. 1), P45OIA1 was found to be expressed in control liver and kidney, though at a 100-fold. lower level than in the liver of   23) preputial gland RNA. The poly(A+) RNA from the lung, intestine, testis, brain, kidney and preputial gland was isolated from Aroclor-treated animals, whereas the lung RNA was from untreated animals; 1.5 ,ug of hepatic poly(A+) RNA from treated animals or 5 ,ug of poly(A+) RNA was used. The gel was subjected to autoradiography for 24 hr. methylcholanthrene-treated animals. By using a dilution series of mRNA from treated animals, we estimate that we should have detected a level of P450IA1 mRNA in the control liver and kidney, which is by a factor of 1000 lower than the level in the induced liver. Our data are supported by a recent studywhich found, using oligomer probes, that only P450IA2, but not P450IA1, is expressed in the liver of untreated animal (11). In the study displayed in Figure 1, the signal consisted of a fully protected fragment, and no distinct partially protected fragments were obtained. However, by using a probe covering the 5' end of the P450IA1 cDNA in the protection assay of RNA isolated from treated animals instead of the P450IA1 (1957-2620) probe, we detected shorter protected fragments in addition to the fully protected probe (data not shown). Only these partially protected fragments were found in the assay of mRNA isolated from untreated animals. We think that the partially protected fragments were derived from the P450IA2 RNA, indicating that only P450IA2 and not P450IA1 is expressed in the liver of untreated animals. Thus our data show, using the highly sensitive RNAse A protection assay, that besides the liver, which expresses P450IA2, no other organ of untreated animals expresses P450IA genes. This observation raises the question whether the corresponding proteins play a role in chemical carcinogenesis, which seems unlikely unless one assumes that at least in humans those proteins are expressed due to exposure to environmental compounds. On the other hand, our study showed (Fig. 2) that members of the P450IIB family, some of which encode the major phenobarbitalinducible cytochromes P-450, are expressed in the lung, the liver, and the preputial gland of untreated animals. However, for untreated animals, only RNA from the lung yielded a considerable amount ofthe fully protected P450IIB1 probe, whereas RNA from the liver and the preputial gland, which is often used as a model for sebaceous glands, yielded only partially protected fragments, indicating that these organs do not express P450IIB1, but other members of the P450IIB family.
Unlike the P450IA family, which appears to be expressed in some extrahepatic tissues of animals that have been treated with the versatile cytochrome P-450inducer Aroclor 1254, and unlike the P4501IB family, which appears to be expressed in some extrahepatic tissues of untreated as well as treated animals, the members of the P450IIC family were found to be expressed only in the liver (Fig. 3). Thus it appears that the corresponding P450IlC proteins do not contribute to the metabolism of carcinogens in extrahepatic tissues.
From our study we conclude that members of the P450IA family might play only a minor role in the extrahepatic metabolism of carcinogens in untreated animals but play an important role in the metabolism of xenobiotics after exposure of the animals to compounds such as Aroclor 1254. In contrast, some members of the P450IIB family seem to be expressed constitutively in some extrahepatic tissues such as the lung and may contribute significantly to the metabolism of xenobiotics in those tissues.