Research Interests of our Laboratory

The cytochromes P450 are ubiquitous proteins, found in just about every phylum. Over 2,000 different forms have been identified. The individual species have large numbers of cytochrome P450 forms. In humans, for example 57 different forms have been identified in the genome. The mouse genome sports 93 different forms. The different forms of cytochrome P450 can be grouped into families, based upon amino acid sequence identity. In mammals Families 1-3 are generally considered as 'xenobiotic-metabolizing' enzymes, oxidizing the greatest number of drugs and chemicals encountered in the environment, as well as a number of endobiotics like steroids destined for excretion. Most of the other families of cytochrome P450 are involved in steroidogenesis or vitamin activiation, or maintenance of homeostasis in the body in one way or another. However, in recent studies it has become apparent that a number of these 'xenobiotic metabolizing' forms of cytochrome P450 are present in the conceptus, the developing embryo and fetus (Choudhary, et al. 2003).

            In earlier studies functional defects in cytochrome P450 1B1 (CYP1B1) was linked to the disease phenotype Primary Congenital Glaucoma (PCG) (Stoilov, et al. 1998). We prepared the cDNA for this form of cytochrome P450, expressed it in Escherichia coli (E. coli) (Jansson, et al. 2000), and examined its properties. While individuals homozygous for truncating mutations in this gene always showed the PCG phenotype, in a number of instances individuals homozygous for mutations in the gene showed incomplete penetrance. Two of these latter mutations appeared to code for intact proteins with just point mutations, G61E and R469W (Fig. 1).

 

 

Figure 1. Comparison of spectra of R469W and G61E with wild type CYP1B1. (Jansson, et al. 2001)

 

. We prepared the cDNA for these mutations, expressed them in E. coli, and examined their properties (Jansson, et al. 2001). The two proteins were capable of similar metabolic activities as the wild type CYP1B1. However, G61E, with a mutation in the 'hinge region' of the protein, had some alteration in metabolite profile with endogenous steroids like b-estradiol; its specific activity was not too dissimilar to that of the wild type, but the ratio of its ability to generate the 4-hydroxy catechol estrogen relative to the 2-hydroxy metabolite was greatly impaired. In contrast, R469W had only about 30% of the overall activity of the wild type and a lesser change in metabolite pattern (Table 1).

_________________________________________

TABLE 1

Steroid Metabolism by membrane CYP1B1 and mutants*

 

Testosterone Metabolism
Metabolite	CYP1B1	G61E	R469W
6b-OH	0.119	0.024	0.026
16a-OH	0.075	0.029	0.029
15a-OH	0.015	0.008	0.007
Total	0.209	0.061	0.062
Progesterone Metabolism
6b-OH	1.02	0.44	0.38
16a-OH	1.57	0.80	0.55
Total	2.59	1.24	0.93
Estradiol Metabolism
4-OH	0.819±0.036	0.267±0.111	0.157±0.076
2-OH	0.324±0.049	0.241±0.71	0.109±0.033
6a-OH	0.079±0.033	0.053±0.008	0.030±0.003
6ß-OH	0.053±0.033	0.052±0.021	0.021±0.009
16a-OH	0.056±0.023	0.038±0.008	0.019±0.004
15a-OH	0.131±0.017	0.115±0.008	0.047±0.007
Total	1.462±0.081	0.767±0.119	0.382±0.070
Ratio 4OH/2OH	2.5	1.1	1.4

­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

*nmol/min/nmol P450. Data are means of 2 experiments except for estradiol data, which are means±SD, n=3. All assays contain bacterial membranes at 0.15 mM cytochrome P450, 0.4 mM NADPH-cytochrome P450 reductase (incorporated in the presence of 0.125% cholate) in 50 mM Na phosphate, pH 7.5, 10 mM MgCl₂, 0.01% Na cholate (final concentration) and either 124 mM testosterone or progesterone, or 62 mM 17b estradiol and 0.2 mCi of ¹⁴C-labelled steroid. Incubations were for 12 minutes at 37^o C after addition of 0.5 mM NADPH. (Jansson, et al. 2001)

_____________________________

 

The spectra of the reduced, carbon monoxide complexes of the purified forms indicated a lower amount of the 450 nm holoenzyme was present in the preparation. When we examined the stability of the proteins it turned out that G61E had a much lower stability, degrading by 50% at 4^o C (Fig 2). In contrast, R469W was as stable as the wild type enzyme. Based upon these observations we suggested that the incomplete penetration might be the result of in utero induction of the defective enzymes to levels sufficient for normal eye development (Jansson, et al. 2001).

 

 

Figure 3. Time course of degradation of CYP1B1 and mutants at 4^o. (Jansson, et al. 2001)

 

The data, thus possibly explains why individuals homozygous for either of these mutations in CYP1B1 show the PCG phenotype, despite producing the holoprotein; lesser amounts of intact holoenzymes are formed, G61E has a lower stability, and both have lower specific activities with endogenous substrates. These mutations result in incomplete penetrance, a number of individuals homozygous for the mutations do not develop the disease phenotype. It is possible that their escape may be related to the inducible nature of the CYP1B1 on challenge with polycyclic aromatic hydrocarbons and other xenobiotics; exposure in utero to such agents would elevate the level of the enzymes, and the amount present might be sufficient for the necessary metabolic function in normal eye development.

 

From our studies it would appear that CYP1B1 is necessary for normal development of the trabecular meshword (Fig. 4). For normal eye development the cells that make up the trabecular meshwork must migrate upward to abut on the anterior chamber where they will serve to filter and drain the anterior chamber. In the absence of the proper migration excessive fluid results in increased pressure that damages the retina. Human CYP1B1 is not the only form of cytochrome P450 known to be involved in normal in utero development. The absence of the orthologous enzyme in mouse, Cyp1b1, as in knockout mice, results in similar inability of the trabecular meshwork to develop normally (Libby, et al. 2003). The difference in the shape of the mouse eye, however, allows the animal to

 

Figure 4. The trabecular meshwork in normal and abnormal eye development. (Stoilov, et al. 2001)

 

develop normal anterior chamber pressure. From such studies we were led to an examination of the role of cytochrome P450 forms in development in the different phyla (Schenkman, et al. 2003). In chickens and rodents a form of cytochrome P450, CYP26, has been shown to be necessary for retinoic acid elimination (Fig. 5). Retinoic acid is an important morphogen, necessary for normal embryo development. A number of teratologies and in utero death occur if retinoic acid homeostasis is disrupted. Deforming mutations in a number of other phyla have been revealed to be the results of mutations in different forms of cytochrome P450. From such observations it is now clear that the

 

Figure 5. Involvement of cytochrome P450 in development in different phyla. (Schenkman, et al. 2003)

 

cytochromes P450 may also function in development of the organism. As a result, research in this laboratory has turned to an examination of the forms of cytochrome P450 present in utero, and how such forms of cytochrome P450 function in development. From the fact that these enzymes are monooxygenases, their role(s) are expected to be the generation of some morphogen necessary for signaling in development of the embryo or tissues. Alternatively, they may function, like CYP26 (Fig 6, orange), in elimination of the function of some active morphogen, preventing its signaling capacity from dispersing beyond the specific cells upon which it must act. Since mouse has a number of orthologous forms of cytochrome P450 present in the human, we turned to the mouse for a model system. In our recent report, we searched mouse embryo cDNA libraries for forms of cytochrome P450. Using 40 different primer pairs we were able to identify 27 different forms of cytochrome P450 cDNA in four different developmental stages (Fig. 6). Some of the

 

Figure 6. Forms of cytochrome P450 at four developmental stages in mouse.

 

Interesting features of the figure include the appearance of Cyp1a1 only at day 7 post conception. It is absent in all subsequent developmental stages through day 17. However, another Family 1 cytochrome P450, Cyp1b1 (red) appears after day 11, and remains through day 17. This is the form so necessary in normal eye development. Generally the Family 1, Family 2 and Family 3 forms of cytochrome P450 have been considered as 'drug metabolizing' forms of the enzyme. From these results (Choudhary, et al. 2003), it is clear that many of the so-called drug metabolizing cytochrome P450s may have developmental function. By the same token, since these forms do metabolize xenobiotics, the in utero exposure to an inducing chemical or inhibitory chemical might be expected to result in interference with the function of such interacting forms of cytochrome P450, and result in teratologies. A current goal of the Schenkman laboratory is to determine the developmental roles of the different forms of cytochrome P450 that appear in utero.

 

Periodically suggestions have appeared in the literature that the enzymes of drug metabolism might have constitutive roles in growth, morphogenesis and/or homeostasis in the body, in addition to their roles in xenobiotic metabolism. The appearance of a number of these forms with known functions in homeostasis in the developing mouse are shown in Figure 6, blue). If forms of cytochrome P450 exist with developmental function, they would be expected to be highly conserved over evolution. In fact, a number of such orthologous forms of cytochrome P450 are found, eight of which are in xenobiotic-metabolizing enzyme Families 1 and 2 (none are found in Family 3) (Choudhary, et al, 2005) . It is notable that these latter orthologs have retained extremely high degrees of sequence identity with evolution. For example CYP1B1 of mouse and rat are about 80% identical in sequence with the human ortholog, and if one examines the sequence identities within the 6 substrate recognition sites (SRS) the values approach 100%  (Choudhary, et al, 2004). One might expect that, since the CYPs are monooxygenases, that the roles of the orthologs in development might involve formation of some critical metabolite or removal of some developmentally active compound at critical stages of development. We have searched for some endobiotic that fits this criteria, testing mouse and human CYP1B1 orthologs for metabolism of steroids, the fatty acid arachidonate, and vitamin A (Choudhary, et al, 2004a) . Arachidonic acid metabolism by CYP1B1, the human enzyme, was moderately good, producing a metabolite pattern distinctly different from two other Family 1 forms, CYP1A1 and CYP1A2. However, since the mouse ortholog, Cyp1b1, did not metabolize this substrate to any extent at physiological concentrations, it was clear that production of a metabolite from this substrate is not be the conserved function of the enzyme in development (Choudhary, et al, 2004a) . Similarly, metabolism of progesterone, testosterone and ß-estradiol proved disappointing. However, both human and mouse CYP1B1 orthologs were able to metabolize vitamin A (retinol) to retinal, and retinal to all-trans retinoic acid, a developmentally active morphogen. Neither were able to metabolize retinoic acid, indicating if this was the conserved function of this form of cytochrome P450, then its function was in production of a developmentally active compound, rather than removal such a compound (Choudhary, et al, 2004a) .

At present this laboratory is examining the temporal and spatial appearance of Cyp1b1 in the developing mouse eye structures by histological and immunohistological methods, in order to gain an understanding of how the absence of a functionally active form of CYP1B1 results in the primary congenital glaucoma (PCG) phenotype. According to the UniGene EST Profile Viewer (http://www.ncbi.nlm.nih.gov/UniGene/UGOrg.cgi?TAXID=10090 ) the are 12 forms of cytochrome p450 expressed in the mouse eye. These include Cyp1b1, Cyp2b10, CYP2b13, Cyp2c29, Cyp2d22, Cyp2w1, Cyp3a13, Cyp3a25, as well as Cyp2f2, Cyp2g1, Cyp2j6 and Cyp2j13. Transcript frequencies (number of copies per 10⁶ transcripts) for these genes is less than 10 in the eye, except for Cyp1b1, which is some 7-fold higher, about 75. Similarly, a considerably higher level of expression of CYP1B1 is found in human eye than other forms of cytochrome P450. We have observed that the enzyme protein is expressed in discrete areas of the mouse eye (Choudhary, et al., 2006) (Figure 7). A paper showing  appearance of Cyp1b1 with

 

 

Figure 7. VIP-stained eye of 28 day old mouse showing regions of staining of Cyp1b1. Letters indicate: cornea, C; anterior chamber, AC; iris, I; lens, L; retina, R; ciliary body, CB; iris, I; irideocorrneal angle, IC; retina, R; inner nuclear layer of retina, IN; outer nuclear layer of retina, ON; retinal pigmented epithelium, RPE; retinal gangllion cells, GC. Punctate staining is seen in the GC and in the IN, but diffuse staining is apparant in the corneal epithelial cells (Choudhary, et al, 2006).

 

ontogeny in the mouse is currently in press (Choudhary, et al, 2007), but can be viewed online at the Drug Metabolism & Disposition site http://dmd.aspetjournals.org/papbyrecent.shtml . The study shows the appearance of the enzyme as early as 15.5 days post conception (E15.5). The enzyme localizes in three distinct regions of the eye; the corneal epithelial cells, the anterior chamber region and the retinal region. It is suggested that the roles of Cyp1b1 in the three regions may differ. In the anterior chamber region it may act to facilitate development of trabecular meshwork in the iridiocorneal angle of the eye, a meshwork of cells and stroma believed to function as a filter for the anterior chamber fluids that are formed in the inner  epithelial cells of the ciliary body. In the retina the enzyme is found in the ganclion cells and in the inner nuclear layer of cells, where it is suggested to function in maintenance of the visual pigment levels. In the cornea it is present in the outermost layer, the epithelial cells, which bring the eye in contact with the environment.In this location the enzyme may make use of its ability to metabolize and detoxify xenobiotics to which the eye is exposed.

 

 

References

 

Choudhary D, Jansson I, Schenkman J B, Sarfarazi M and Stoilov I 2003 Comparative expression profiling of 40 mouse cytochrome P450 genes in embryonic and adult tissues.; Arch. Biochem. Biophys. 414 91-100.

 

Choudhary, D.; Jansson, I.; Sarfarazi, M.; Schenkman, J. B. 2004 Xenobiotic-metabolizing cytochromes P450 in ontogeny: evolving perspective. Drug Metab. Rev. ; 36: 547-566.

Choudhary, D.; Jansson, I.; Stoilov, I.; Sarfarazi, M.; Schenkman, J. B. 2004a Metabolism of retinoids and arachidonic acid by human and mouse cytochrome P450 1B1. Drug Metab. Dispos. ; 32: 1-8.

Choudhary, D.; Jansson, I.; Stoilov, I.; Sarfarazi, M.; Schenkman, J. B. 2005 Expression patterns of mouse and human CYP orthologs (families 1-4) during development and in different adult tissues Arch. Biochem. Biophys. ; 436: 50-61.

Choudhary D, Jansson I, Sarfarazi M and Schenkman JB 2006  Physiological significance and expression of P450s in the developing eye. Drug Metab. Rev. 38:337-352.

 Jansson I, Stoilov I, Sarfarazi M and Schenkman J B 2000 Enhanced expression of CYP1B1 in Escherichia coli.; Toxicology 144 211-219.

 

Jansson I, Stoilov I, Sarfarazi M and Schenkman J B 2001 Effect of two mutations of human CYP1B1, G61E and R469W, on stability and endogenous steroid substrate metabolism.; Pharmacogenetics 11 1-9.

 

Libby R T, Smith R S, Savinova O V, Zabaleta A, Martin J E, Gonzalez F J and John S W 2003 Modification of ocular defects in mouse developmental glaucoma models by tyrosinase; Science 299 1578-1581.

 

Schenkman J B, Choudhary D, Jansson I, Sarfarazi M and Stoilov I 2003 Involvement of cytochromes P450 in development.; Proc. Indian Natn. Sci. Acad. 2003; B69: 917-929.

 

Stoilov I, Akarsu A N, Alozie I, Child A, Barsoum-Homsy M, Turacli M E, Or M, Lewis R A, Ozdemir N, Brice G, Aktan S G, Chevrette L, Coca-Prados M and Sarfarazi M 1998 Sequence analysis and homology modeling suggest that primary congenital glaucoma on 2p21 results from mutations disrupting either the hinge region or the conserved core structures of cytochrome P4501B1.; Am. J. Hum. Genet. 62 573-584.

 

Stoilov, I.; Jansson, I.; Sarfarazi, M.; Schenkman, J. B. 2001 Roles of cytochrome P450 in development.; Drug Metab. Drug Interac. 18: 33-55.