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Human CYP4B1, a cytochrome P450 monooxygenase predominantly expressed in the lung, inefficiently metabolizes classical CYP4B1 substrates, such as the naturally occurring furan pro-toxin 4-ipomeanol (4-IPO). Highly active animal forms of the enzyme convert 4-IPO to reactive alkylating metabolite(s) that bind(s) to cellular macromolecules. By substitution of 13 amino acids, we restored the enzymatic activity of human CYP4B1 toward 4-IPO and this modified cDNA is potentially valuable as a suicide gene for adoptive T-cell therapies. In order to find novel pro-toxins, we tested numerous furan analogs in in vitro cell culture cytotoxicity assays by expressing the wild-type rabbit and variants of human CYP4B1 in human liver-derived HepG2 cells. To evaluate the CYP4B1 substrate specificities and furan analog catalysis, we optimized the N-terminal sequence of the CYP4B1 variants by modification/truncation and established their heterologous expression in Escherichia coli (yielding 70 and 800 nmol·l-1 of recombinant human and rabbit enzyme, respectively). Finally, spectral binding affinities and oxidative metabolism of the furan analogs by the purified recombinant CYP4B1 variants were analyzed: the naturally occurring perilla ketone was found to be the tightest binder to CYP4B1, but also the analog that was most extensively metabolized by oxidative processes to numerous non-reactive reaction products., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

We developed a weak-affinity separation system for single-nucleotide polymorphisms (SNPs) based on capillary electrophoresis. In this approach, single-stranded DNA (ssDNA)-polyacrylamide (polyAAm) conjugate was used as a pseudo-immobilized affinity ligand to separate the target DNA, cytochrome P450 2C9 (CYP2C9), and its point mutant. The ligand DNA was designed to be complementary to the normal DNA, and the target DNA was electrophoretically separated by the difference in the affinity with the pseudo-immobilized ligand in the capillary. We showed that the separation efficiency was closely associated with the Tm value of double-stranded DNA (dsDNA) consisting of the target and ligand DNA, which depends on the measurement conditions, such as the base number of the ligand DNA and the concentration of Mg2+ in the buffer solution.

Mouse skin contains a NADPH-dependent, aryl hydrocarbon hydroxylase (AHH), which is inducible by polycyclic aromatic hydrocarbons. In general, unsubstituted polycyclic hydrocarbons caused a greater induction of epidermal AHH than substituted one (1,2,3,4-dibenzanthracene greater than 1,2,5,6-dibenzanthracene greater than benz (a)anthracene equal 3-methylcholanthrene greater than 7,12-dimethlbenz (a)anthracene) which did not correlate with their ability to initiate tumors in mouse skin. Two different techniques were used to isolate epidermis and similar results were obtained with both. However, the technique of isolating epidermis using a mild heat treatment required that the temperature be maintained at 52 degrees C for 30 sec. If the temperature was raised to 54 degrees C or above, there was a large reduction in the AHH activity. Isolated epidermis has 4 to 5 times the AHH activity as dermis and about twice that of whole skin. This was true for control mice or mice in which AHH was induced by pretreatment with benz(a)anthracene.