Areas of Interest
The cytochrome P450s are ubiquitous proteins found in most
phyla. Several thousand different forms have already been
identified. The individual species have large numbers of
cytochrome P450 forms. In humans, for example, 57 different
forms of cytochrome P450 have been identified in the genome. The
mouse genome contains 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 or used in intermediary metabolism of
compounds necessary for homeostasis. Most of the families of
cytochrome P450 are involved, in one way or another, in
steroidogenesis, vitamin metabolism or maintenance of
homeostasis in the body. In recent studies, however, it has
become apparent that a number of the xenobiotic metabolizing
cytochrome P450s are present in the conceptus and the developing
embryo and fetus at specific times after conception (Choudhary
et al., 2004a; Choudhary et al., 2003; Choudhary et al., 2005),
appearing to function as development-influencing gene products (Schenkman
et al., 2003).
Mutations in the cytochrome P450 1B1 (CYP1B1) gene in humans
results in the disease, Primary Congenital Glaucoma (PCG), which
appears to be due to abnormal development of the trabecular
meshwork at the angle of the eye. Although the mouse does not
develop the same disease phenotype, damage to the mouse
orthologous gene does result in similar histologically observed
abnormal development of the trabecular meshwork. We have shown
that point mutations in the human gene, which in patients cause
PCG, result in forms of CYP1B1 that are expressed as a
holoenzyme, with lesser stability and lower specific activity
toward endobiotics (Choudhary et al., 2004b; Jansson et al.,
2001). We have used the mouse to examine the temporal and
spatial appearance of Cyp1b1 (the mouse ortholog) protein in
mouse eye structures by histological and immunohistological
methods (Choudhary et al., 2006) in order to gain an
understanding of how the absence of a fully active form of the
enzyme might result in the PCG phenotype. We note that, "the
distinctive constitutive expression pattern of some CYPs during
mouse ontogeny and their presence in human fetal tissues
suggests an involvement in morphogenesis and patterning of
different embryonic structures. This might occur by modulating
the level of ligands required for activation of membrane or
nuclear receptors", and it was suggested that such ligands were
probably endobiotics of lipophilic properties (Choudhary et al.,
2006). Examination of the effects of four different PCG-causing
mutations of CYP1B1 on the stability and catalytic activities
revealed impairment of these characteristics by the mutations,
strongly suggestion diminution of function in the absence of
enhanced levels of the enzyme are the cause of the observed PCG
phenotype (Choudhary et al., 2008). Toward this observation, it
was suggested that CYP1B1 might eventually serve as a potential
target for gene therapy of the PCG condition (Choudhary et al.,
2009).
Metabolism of different chemicals by cytochrome P450 forms
often results in their conversion into active electrophiles that
can react with DNA. It is generally accepted that such reactions
are the cause of different cancers. In other studies we have
been examining the ability to make use of cytochrome P450 forms
in electrochemical analysis of the potential of different drugs
and environmental pollutants to cause genotoxic effects by
virtue of reacting with the DNA in thin films (Estavillo et al.,
2003; Munge et al., 2003; Rusling et al., 2008; Zhou et al.,
2003). Using microsomes containing cytochrome P450 and
cytochrome P450 reductase (CPR) we have been able to show the
involvement of CPR in the electrochemical activation of these
environmental pollutants by cytochrome P450 forms (Sultana et
al., 2005; Sultana et al., 2007). In attempts to make rapid
throughput of analyses, arrays have been developed in which
multiple forms of activating enzyme could be inserted to take
advantage of differences in substrate specificities (Wang et
al., 2005). Using the electrochemical method and layer-by-layer
formation of interacting cytochrome P450 system components and
DNA it could be shown that reactive intermediates were formed
which could complex with and damage the DNA (Krishnan et al.,
2007; So et al., 2008; So et al., 2007). Arrays for the
electrochemical activation of the carcinogen benzo(a)pyrene by
multiple forms of cytochrome P450 were developed (Hvastkovs et
al., 2007). One could also use microsomes containing mixtures of
metabolic enzymes in thin films with DNA for screening compounds
for genotoxicity (Krishnan et al., 2008), or pure enzymes,
developing methods for screening the DNA (Bajrami et al., 2008;
Fundador et al., 2008). We have been monitoring DNA reactivity
with different potential toxicants (Bajrami et al., 2009; Hull
et al., 2009; Krishnan et al., 2009; Zhao et al., 2009a; Zhao et
al., 2009b).
Publications
Selected Publications
Krishnan S, Hvastkovs EG, Bajrami B, Schenkman JB and Rusling
JF (2009) Human cyt P450 mediated metabolic toxicity of
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) evaluated
using electrochemiluminescent arrays. Mol Biosyst 5(2):163-169.
Hull DO, Bajrami B, Jansson I, Schenkman JB and Rusling JF
(2009) Characterizing metabolic inhibition using electrochemical
enzyme/DNA biosensors. Anal Chem 81(2):716-724.
Choudhary D, Jansson I and Schenkman JB (2009) CYP1B1, a
developmental gene with a potential role in glaucoma therapy.
Xenobiotica 39:606-615.
Bajrami B, Zhao L, Schenkman JB and Rusling JF (2009) Rapid
LC-MS drug metabolite profiling using microsomal enzyme
bioreactors in a parallel processing format. Anal Chem
81(24):9921-9929.
Zhao L, Krishnan S, Zhang Y, Schenkman JB and Rusling JF
(2009a) Differences in metabolite-mediated toxicity of tamoxifen
in rodents versus humans elucidated with DNA/microsome
electro-optical arrays and nanoreactors. Chem Res Toxicol
22(2):341-347.
Zhao L, Schenkman JB and Rusling JF (2009b) Screening for
reactive metabolites using electro-optical arrays featuring
human liver cytosol and microsomal enzyme sources and DNA. Chem
Commun (Camb)(36):5386-5388.
Choudhary D, Jansson I, Sarfarazi M and Schenkman JB (2008)
Characterization of the biochemical and structural phenotypes of
four CYP1B1 mutations observed in individuals with primary
congenital glaucoma. Pharmacogenetics and Genomics
18(8):665-676.
Bajrami B, Hvastkovs EG, Jensen GC, Schenkman JB and Rusling
JF (2008) Enzyme-DNA biocolloids for DNA adduct and reactive
metabolite detection by chromatography-mass spectrometry. Anal
Chem 80(4):922-932.
Fundador EV, Choudhary D, Schenkman JB and Rusling JF (2008)
Accurate DNA fragment sizing by capillary electrophoresis with
laser-induced fluorescence array for detection of sequence
specificity of DNA damage. Anal Chem 80(6):2212-2221.
Krishnan S, Hvastkovs EG, Bajrami B, Choudhary D, Schenkman
JB and Rusling JF (2008) Synergistic metabolic toxicity
screening using microsome/DNA electrochemiluminescent arrays and
nanoreactors. Anal Chem 80(14):5279-5285.
Rusling JF, Hvastkovs EG, Hull DO and Schenkman JB (2008)
Biochemical applications of ultrathin films of enzymes, polyions
and DNA. Chem Commun (Camb)(2):141-154.
So M, Hvastkovs EG, Bajrami B, Schenkman JB and Rusling JF
(2008) Electrochemical genotoxicity screening for arylamines
bioactivated by N-acetyltransferase. Anal Chem 80(4):1192-1200.
So M, Hvastkovs EG, Schenkman JB and Rusling JF (2007)
Electrochemiluminescent/voltammetric toxicity screening sensor
using enzyme-generated DNA damage. Biosens Bioelectron
23(4):492-498.
Hvastkovs EG, So M, Krishnan S, Bajrami B, Tarun M, Jansson
I, Schenkman JB and Rusling JF (2007) Electrochemiluminescent
arrays for cytochrome P450-activated genotoxicity screening. DNA
damage from benzo[a]pyrene metabolites. Anal Chem
79(5):1897-1906.
Krishnan S, Hvastkovs EG, Bajrami B, Jansson I, Schenkman JB
and Rusling JF (2007) Genotoxicity screening for N-nitroso
compounds. Electrochemical and electrochemiluminescent detection
of human enzyme-generated DNA damage from N-nitrosopyrrolidine.
Chem Commun (Camb)(17):1713-1715.
Sultana N, Schenkman JB and Rusling JF (2007) Direct
electrochemistry of cytochrome P450 reductases in surfactant and
polyin films. Electroanalysis 19:2499-2506.
Choudhary D, Jansson I, Sarfarazi M and Schenkman JB (2006)
Physiological significance and expression of P450s in the
developing eye. Drug Metabolism Reviews 38(1-2):337-352.
Choudhary D, Jansson I, Stoilov I, Sarfarazi M and Schenkman
JB (2005) Expression patterns of mouse and human CYP orthologs
(families 1-4) during development and in different adult
tissues. Archives Biochemistry Biophysics 436(1):50-61.
Wang B, Jansson I, Schenkman JB and Rusling JF (2005)
Evaluating enzymes that generate genotoxic benzo[a]pyrene
metabolites using sensor arrays. Anal Chem 77(5):1361-1367.
Sultana N, Schenkman JB and Rusling JF (2005) Protein film
electrochemistry of microsomes genetically enriched in human
cytochrome p450 monooxygenases. J Am Chem Soc
127(39):13460-13461.
 View more publications, see
Pubmed listing.
rev. 7/10 |
