Letter

Top Letter References

In the June issue (29:908-915) of Drug Metabolism and Disposition, an interesting article on hepatic cytochromes P450 in pig was published by Myers et al. (2001). The authors looked for swine cytochromes P450 (P450¹) analogous to human or rat liver microsomal P450 enzymes using Western blotting. In the next step, an induction of P450 enzymes had been followed after induction by a mixture of popular inducers, such as -naphthoflavone, phenobarbital, and dexamethasone, to confirm the expected induction of CYP1A, 2A, B, and C, and 3A activities. From differences in specific activities expressed for characteristic substrates of various P450 enzymes present in microsomes or S9 fraction, the authors concluded that there are many similarities, but also some differences, between pig and human cytochrome P450 enzymes. The conclusion reached by the authors is correct; however, we feel that it is necessary to comment on several points raised in the article in question.

First, the fact that several pig P450 enzymes have been cloned and characterized (i.e., not only those mentioned by authors as steroid-metabolizing ones) is overlooked. CYP3A29 has been identified in porcine small intestine, and two more cDNA clones belonging to the CYP2C subfamily were also characterized from the same source (Nissen et al., 1998). Because it is well known that the P450 enzymes of the gastrointestinal tract are identical, the pig CYP3A29 of the small intestine and this of the liver are the same gene products. An interesting conclusion on the presence of CYP2D enzymes in pig liver microsomes was obtained recently; a CYP2D enzyme, performing N-hydroxylations and N-reductions, was isolated and characterized (Clement et al., 1997). This enzyme has been shown to catalyze N-demethylation of dextromethorphan, a prototypical reaction of human CYP2D6. Moreover, pig liver microsomal vitamin D₃ hydroxylating CYP2D25 has been isolated and thoroughly characterized (Hosseinpour and Wikvall, 2000). This enzyme, showing 77% sequence identity with human CYP2D6, is able to share some substrate specificities with the human enzyme. Hence, the statement of the authors (Myers et al., 2001) that "... the available information on DNA sequences of swine P450 enzymes is confined to those enzymes responsible for steroid metabolism in the ovary, brain, and adrenal gland" does not give the full picture of the actual progress in this field.

The second comment deals with the investigation of induction of various pig hepatic P450 enzymes by different inducers. First of all, the methodical approach chosen by the authors was not able to give detailed information on the induction of particular P450 activity because the inductors were given to two identical animals in a sequence of 3 days. The animals were induced with the phenobarbital on day 1, then with -naphthoflavone on days 2 and 3, and finally with dexamethasone again on day 3. This approach, however, excluded the possibility to discern the effect of phenobarbital from that of the dexamethasone. In fact, there are numerous detailed studies on the influence of various inducers to liver microsomal P450 activities in the pig or minipig providing information both on the activities and the relative amounts of particular P450 enzymes by Western blotting. For example, the seminal work of Monshouwer et al. (1998) documenting the inducibility of CYP1A, 2C, and 3A by -naphthoflavone, phenobarbital, and rifampicin gave practically the same results as presented here. Also, more recent works of Skaanild and Friis (1999, 2000) document the presence of individual activities and expression of respective P450 enzymes in pig or minipig liver. The induction protocol used by Myers et al. (2001) unfortunately was not able to contribute to the discussion on the inducibility of pig CYP3A by dexamethasone in microsomes and hepatocytes; although Monshouwer et al. (1998) were able to detect some induction of this form, Lu and Li (2001) failed to detect any induction of the CYP3A activity in pig hepatocytes by this compound.

The third and last comment is more general. Possibly, the conclusions on the inducibility and properties of particular pig P450 enzymes should be taken with some caution. For example, the CYP2D6 form is known not to be inducible at all, and the CYP2E1 form is known to be induced by other inducers that were tested in this work (Guengerich, 1995). The substrate specificity of the CYP2E1 must be tested in a reconstituted system with an isolated protein; until then, the conclusions on its specificity are more or less suggestions. Also, the antibodies used for Western blotting should be anti-human rather than (in the majority) anti-rat or rabbit, as it has been used in works of Skaanild and Friis (1999, 2000) or in our article on this subject (Anzenbacher et al., 1998)

Hence, the article by Myers et al. (2001) opens some interesting questions and contributes to the general knowledge of the pig (minipig) liver microsomal P450 enzymes. Because the (mini)pig is a purported source of cells, tissues, and organs in human therapy (see Levy et al., 2000 or Dixit and Gitnick, 1996 for a review) and, possibly, a species of choice for studies of hepatic metabolism of drugs in humans (Anzenbacher et al., 1998), these studies are needed.

	Response to Letter to the Editor

We would like to take advantage of the opportunity that the letter from Dr. Anzenbacher and colleagues presents to re-emphasize the key point of our article (Myers et al., 2001), namely that one cannot universally ascribe identical behavior to the same P450¹ isozyme in a different species just because it cross-reacts with antibodies. In other words, it is more appropriate to actually conduct the research in the target species rather than use a surrogate model system.

We appreciate the information provided by Dr. Anzenbacher and colleagues concerning the genetic identification of swine P450 (Nissen et al., 1998; Hosseinpour and Wikvall, 2000). However, it is important to correct some omissions from their letter and statements concerning the interpretation of the data in those two articles. As will be shown below, there is, in reality, compelling genetic evidence for only two complete functional swine cytochromes P450 and not the four suggested by Dr. Anzenbacher and colleagues. Thus, the statement from our article is still correct, namely that there is a paucity of information on the identity of swine cytochrome P450 at the genetic level when compared with the vast amount of information available for humans and rodents.

Careful analysis of the article by Nissen et al. (1998) demonstrates that only one complete swine P450 gene was reported, CYP3A29. The swine 2C genes reported in Nissen's article (CYP2C42 and CYP2C42P1) represent a gene with a truncated 5' end (CYP2C42) and a pseudogene (CYP2C42P1). CYP2C42 and CYP2C42P1 differ from each other by only five nucleotides, clearly indicating that these two were derived from a common ancestor. CYP2C42 encodes an open reading frame of only 327 amino acids. As the article itself clearly states, "comparison with other genes from the 2C family indicate that CYP2C42 is not a full-length clone". Thus, neither of these two genes represents a functional enzyme.

This is not the only study looking at swine 2C. Using a reverse transcription-polymerase chain reaction approach, Zaphiropoulos et al. (1995) identified five cDNA clones from preovulatory follicles and six from the corpus luteum. After DNA sequencing of the resulting amplicons, designations of CYP2C32, CYP2C33, CYP2C34, CYP2C35, and CYP2C36 were assigned to those amplicons demonstrating greater than 3% divergence. This criteria was established by the P450 Nomenclature Committee to distinguish between alleles and unique P450s. Using this criteria, Zaphiropoulos and colleagues identified two alleles for CYP2C33 and four alleles for CYP2C34. However, as none of the amplicons resulted in identification of full-length genes, it is unknown whether any or all are functional genes. The results of their study suggest the presence of at least six different isoforms in porcine ovary. However, based on these two studies, the issue of swine 2C functionality is still unknown. From work still in progress in our laboratory, we have tentatively identified four swine hepatic 2C isoforms, based on immunochemical reactivity.

However, the issue of functionality of one of these 2C genes may have been addressed. In work looking at endothelium-derived hyperpolarizing factor (EDFH) in porcine coronary artery epithelial cells (Fisslthaler et al., 1999), addition of -naphthoflavone enhanced the formation of 11,12-epoxyeicosatriene acid. -Naphthoflavone also enhanced EDFH-mediated hyperpolarization and relaxation. Adding CYP2C34 antisense oligonucleotides decreased the level of CYP2C34 protein. These results suggest that at least one of the 2C genes identified by Zaphiropoulos et al. (1995) is functional.

The statements by Dr. Anzenbacher and colleagues concerning swine 2D25 imply that the substrate specificities were determined by Hosseinpour and Wikvall (2000) as identical to human 2D6. Although swine 2D25 catalyzed conversion of tolterodine (a 2D6 substrate) to its 5-hydroxymethyl metabolite, human 2D6 did not catalyze the 25-hydroxylation of vitamin D, as did swine. Purified swine 2D metabolizes benzamidoxime (a 2D6 substrate) to benzamide (Clement et al., 1997). Our own results demonstrated that swine microsomes are capable of metabolizing propranolol, bufuralol, and dextromethorphan, classic 2D6 substrates (Myers et al., 2001). Thus, swine 2D25 is capable of metabolizing some of the same substrates associated with human 2D6, whereas human 2D6 cannot catalyze the same substrates as swine 2D25. Clearly, the 23% difference in structure that Dr. Anzenbacher and colleagues dismiss as trivial confers some significant differences.

Throughout their letter, Dr. Anzenbacher and colleagues freely interchange work conducted with minipigs with work done in domestic pigs. It is obvious that Dr. Anzenbacher's contention is that minipigs and domestic swine are identical, when in fact they are not. Their own work (Anzenbacher et al., 1998) clearly demonstrates this fact. Their work with microsomes from Göttingen minipigs shows that these animals may contain one of two different 3A isoforms. The results from our study with domestic swine demonstrated one constitutive isoform and one inducible isoform. In work not yet published, we demonstrate the presence of the same constitutive isoform in liver microsomes obtained from 33 normal animals. This finding differs from that of Hosagrahara and colleagues (1999), who demonstrated the presence of at least two, and possibly four, 3A isoforms in livers obtained from Dorac pigs. The pigs we have examined have all been Landrace-Poland China crossbred animals. Therefore, the breed of animal may play a role in the difference. Other differences may lie in the choice of antisera. We used commercially available anti-rat antisera, whereas Hosagrahara and colleagues (1999) used privately prepared anti-human 3A antisera. In addition, the Göttingen minipigs have no 2D6 activity (Skaanild and Friis, 1999). The results presented in our article (Myers et al., 2001) clearly demonstrated that domestic swine have the capacity to metabolize substrates associated with 2D6 and a 2D6-like immunoreactive protein. These results are in agreement with those of Jurima-Romet et al. (2000), who demonstrated the presence of a 2D-like molecule in domestic swine liver microsomes and hepatocytes using 2D6-specific substrates, inhibitors of 2D6 activity and anti-2D6 antibodies.

Further confounding the minipig issue is the fact there are numerous breeds of minipigs, none of which are universally available. The Göttingen minipigs, used in the studies cited by Dr. Anzenbacher and colleagues, are only available in Europe, whereas the Hormel, Pittmoore, Hanford, and Yucatan minipigs are the breeds available in North America. The Yucatan pig is unique in that it is the only naturally occurring minipig. The Ohmini minipig is the primary strain used in Japan.

Several investigators studying minipigs have proposed minipigs as a model for human metabolic studies based on their capacity to metabolize substrates associated with human P450 isoforms. However, a recent study by Lu and Li (2001) using cultured hepatocytes from rats, Yucatan minipigs, dogs, and humans came to the conclusion that the most appropriate model for human preclinical studies was human hepatocytes. Whether the lack of correlation of human and minipig data results from differences between the Yucatan and Göttingen minipigs cannot be determined at this time. However, these possible differences do raise issues that need to be addressed. On the basis of these discrepancies, we would suggest that someone do a comparative study to assess the distribution and enzymatic capabilities of cytochrome P450s in the various minipig strains.

The criticism of our induction protocol demonstrates a lack of understanding of the goals of this study (Myers et al., 2001). Nowhere in the article do we claim to be able to discern patterns of induction due to individual agents. Intuitively, one would not expect to be able to make such a claim. The Monshouwer article (1998) failed to demonstrate constitutive expression of 1A2 and 2B proteins in cultured hepatocytes. That work also did not show induction of 2B protein by phenobarbital (PB) based on Western Blot analyses, and the induction of 1A2 by -naphthoflavone (-NF) was marginal. In the article, dexamethasone, PB, and rifampin are stated as inducing 3A protein levels; the figure presented only suggests that PB is inducing 3A protein levels. Using dot-blot hybridization assays, induction of these isoforms was noted; however, data from Northern blot analyses were not presented. Induction by -NF, PB, dexamethasone, or rifampin enhanced the metabolism of testosterone. Rifampin, -NF, and PB induction augmented the metabolism of ethylmorphine (2C substrate), tolbutamide (2C substrate), and caffeine (1A2 substrate). Although these results are intriguing, they differ significantly from what one would predict and also differ from the results of our own work using microsomes from induced pigs. As stated in their letter, these results differ from those of Lu and Li (2001), who showed that in Yucatan minipigs, only rifampin induced 3A expression in cultured hepatocytes. However, the results of Lu and Li (2001) are consistent with those from Hosagrahara et al. (1999) and Hansen et al. (2000); the latter two groups used hepatocytes derived from domestic swine. Both groups showed induction of 3A in cultured hepatocytes from domestic swine and augmented metabolic activities. In addition, all three groups (Hosagrahara et al., 1999; Hansen et al., 2000; Lu and Li, 2001) demonstrated induction of 1A1 protein and metabolic activities, whereas Monshouwer et al. (1998) did not show induction of 1A1.

In conclusion, we agree that much work is needed before the level of understanding of swine cytochrome P450 approaches that available for humans and rodents. This exchange of letters also highlights the difficulties researchers face in trying to maintain scientific currency in the face of innumerable sources of information.