Structure-function relationships for inhibition of human cytochrome P450s (P450s) 1A1, 1A2, 1B1, 2C9, and 3A4 by 33 flavonoid derivatives were studied. Thirty-two of the 33 flavonoids tested produced Reverse Type I binding spectra with P450 1B1, and the potencies of binding were correlated with the abilities to inhibit 7-ethoxyresorufin O-deethylation activity. The presence of a hydroxyl group in flavones, e.g. 3-, 5-, and 7-monohydroxy-and 5,7-dihydroxyflavone, decreased the 50% inhibition concentration (IC 50 ) of P450 1B1 from 0.6 µM to 0.09, 0.21, 0.25, and 0.27 µM, respectively, and 3,5,7-trihydroxyflavone (galangin) was the most potent, with an IC 50 of 0.003 µM. The introduction of a 4'-methoxy-or 3',4'-dimethoxy group into 5,7-dihydroxyflavone yielded other active inhibitors of P450 1B1 with IC 50 values of 0.014 and 0.019 µM, respectively. The above hydroxyl-and/or methoxy-groups in flavone molecules also increased the inhibition activity with P450 1A1 but not always towards P450 1A2, where 3-, 5-, or 7-hydroxyflavone, and 4'-methoxy-5,7-dihydroxyflavone were less inhibitory than flavone itself. P450 2C9 was more inhibited by 7-hydroxy-,5,7-dihydroxy-, and 3,5,7-trihydroxyflavones than by flavone but was weakly inhibited by 3-and 5-hydroxyflavone. Flavone and several other flavonoids produced Type I binding spectra with P450 3A4, but such binding was not always related to the inhibitiory activities towards P450 3A4. These results indicate that there are different mechanisms of inhibition for P450s 1A1, 1A2, 1B1, 2C9, and 3A4 by various flavonoid derivatives and that the number and position of hydroxyl and/or methoxy groups highly influence the inhibitory actions of flavonoids towards these enzymes. Molecular docking studies suggest that there are different mechanisms involved in the interaction of various flavonoids with the active site of P450s, thus causing differences in inhibition of these P450 catalytic activities by flavonoids.

We have analyzed the function of Hansenula polymorpha Pex14p in selective peroxisome degradation. Previously, we showed that Pex14p was involved in peroxisome biogenesis and functions in peroxisome matrix protein import. Evidence for the additional function of HpPex14p in selective peroxisome degradation (pexophagy) came from cells defective in HpPex14p synthesis. The suggestion that the absence of HpPex14p interfered with pexophagy was further analyzed by mutational analysis. These studies indicated that deletions at the C terminus of up to 124 amino acids of HpPex14p did not affect peroxisome degradation. Conversely, short deletions of the N terminus (31 and 64 amino acids, respectively) of the protein fully impaired pexophagy. Peroxisomes present in these cells remained intact for at least 6 h of incubation in the presence of excess glucose, conditions that led to the rapid turnover of the organelles in wild-type control cells. We conclude that the N terminus of HpPex14p contains essential information to control pexophagy in H. polymorpha and thus, that organelle development and turnover converge at Pex14p.

We have cloned the Hansenula polymorpha PEX14 gene by functional complementation of the chemically induced pex14‐1 mutant, which lacked normal peroxisomes. The sequence of the PEX14 gene predicts a novel protein product (Pex14p) of 39 kDa which showed no similarity to any known protein and lacked either of the two known peroxisomal targeting signals. Biochemical and electron microscopical analysis indicated that Pex14p is a component of the peroxisomal membrane. The synthesis of Pex14p is induced by peroxisome‐inducing growth conditions. In cells of both pex14‐1 and a PEX14 disruption mutant, peroxisomal membrane remnants were evident; these contained the H.polymorpha peroxisomal membrane protein Pex3p together with a small amount of the major peroxisomal matrix proteins alcohol oxidase, catalase and dihydroxyacetone synthase, the bulk of which resided in the cytosol. Unexpectedly, overproduction of Pex14p in wild‐type H.polymorpha cells resulted in a peroxisome‐deficient phenotype typified by the presence of numerous small vesicles which lacked matrix proteins; these were localized in the cytosol. Apparently, the stoichiometry of Pex14p relative to one or more other components of the peroxisome biogenesis machinery appears to be critical for protein import.

A total of 68 chemicals including derivatives of naphthalene, phenanthrene, fluoranthene, pyrene, biphenyl, and flavone were examined for their abilities to interact with human P450s 2A13 and 2A6. Fifty-one of these 68 chemicals induced stronger Type I binding spectra (iron low- to high-spin state shift) with P450 2A13 than those seen with P450 2A6, i.e. the spectral binding intensities (ΔAmax/Ks ratio) determined with these chemicals were always higher for P450 2A13. In addition, benzo[c]phenanthrene, fluoranthene, 2,3-dihydroxy-2,3-dihydrofluoranthene, pyrene, 1-hydroxypyrene, 1-nitropyrene, 1-acetylpyrene, 2-acetylpyrene, 2,5,2’,5’-tetrachlorobiphenyl, 7-hydroxyflavone, chrysin, and galangin were found to induce a Type I spectral change only with P450 2A13. Coumarin 7-hydroxylation, catalyzed by P450 2A13, was strongly inhibited by 2’-methoxy-5,7-dihydroxyflavone, 2-ethynylnaphthalene, 2’-methoxyflavone, 2-naphththalene propargyl ether, acenaphthene, acenaphthylene, naphthalene, 1-acetylpyrene, flavanone, chrysin, 3-ethynylphenanthrene, flavone, and 7-hydroxyflavone; these chemicals induced Type I spectral changes with low Ks values. On the basis of the intensities of the spectral changes and inhibition of P450 2A13, we classified the 68 chemicals into eight groups based on the order of affinities for these chemicals and inhibition of P450 2A13. The metabolism of chemicals by P450 2A13 during the assays explained why some of the chemicals that bound well were poor inhibitors of P450 2A13. Finally, we compared the 68 chemicals for their abilities to induce Type I spectral changes of P450 2A13 with the Reverse Type I binding spectra observed with P450 1B1: 45 chemicals interacted with both P450s 2A13 and 1B1, indicating that the two enzymes have some similarty of structural features regarding these chemicals. Molecular docking analyses suggest similarities at the active sites of these P450 enzymes. These results indicate that P450 2A13, as well as Family 1 P450 enzymes, is able to catalyze many detoxication and activation reactions with chemicals of environmental interest.

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Fifty-one chemicals including derivatives of sixteen flavonoids, three stilbenes, six pyrenes, seven naphthalenes, seven phenanthrenes, ten biphenyls, 17β-estradiol, and estrone were examined for their abilities to induce Reverse Type I binding spectra with human cytochrome P450 (P450) 1B1 and to inhibit 7-ethoxyresorufin O-deethylation (EROD) activities catalyzed by P450 1B1. Forty nine chemicals showed Reverse Type I spectra with P450 1B1 and we found that 3,5,7-trihydroxyflavone, 3′,4′-dimethoxy-5,7-dihydroxyflavone, 4′-methoxy-5,7-dihydroxyflavone, α- and β-naphthoflavones, 2,4,3′,5′-tetramethoxystilbene, pyrene, and several acetylenic pyrenes and phenanthrenes were strong inducers of the spectra and also potent inhibitors of EROD activities catalyzed by P450 1B1. Spectral dissociation constant (Ks) and the magnitude of the binding (ΔAmax/Ks) of 49 chemicals were correlated with the inhibition potencies of EROD activities by these chemicals (correlation coefficients (r) of 0.72 and 0.74, respectively). The Ks and ΔAmax/Ks values were more correlated with IC50 values when compared in a group of derivatives of flavonoids, stilbenes, and estrogens (r=0.81 and 0.88, respectively) or a group of derivatives of pyrenes, naphthalenes, phenanthrenes, and biphenyls (r=0.88 and 0.91, respectively). Among 14 flavonoids examined, 3,5,7-trihydroxyflavone and 4′-methoxy- and 3′,4′-dimethoxy-5,7-dihydroxyflavone were more active than flavone in interacting with P450 1B1, but the respective 7,8-dihydroxyflavones were less active. Pyrene itself was highly active in interacting with P450 1B1, but its binding was slightly decreased when substituted with acetylenic groups. In contrast, substitution of naphthalene with methyl- and ethyl propargyl ethers led to more interaction with P450 1B1 than with naphthalene itself. Similarly, substitution on phenanthrene or biphenyl with acetylenic groups and propargyl ethers increased affinities to P450 1B1. These results suggest that the Reverse Type I binding of chemicals to P450 1B1 may determine how they interact with and inhibit the catalytic activity of the enzyme. Substitutions on the compounds with various acetylenic groups and propargyl ethers cause an increase or decrease of their affinities to P450 1B1, depending on the parent compound used.

The developmentally regulated expression of forms of cytochrome P-450, namely, those encoded by lambda HFL33 and NF25 or HLp cDNAs, which were isolated from respective fetal and adult human liver cDNA libraries, was investigated. When EcoRI fragments of cDNA clones of lambda HFL33 and NF25 were used as probes, these probes hybridized with RNA from both fetal and adult human livers. However, when oligonucleotides specific to the coding and 3'-noncoding region of lambda HFL33 (oli-HFL and oli-HFL3', respectively) were used as probes, these probes gave hybridizable bands with RNA from fetal but not adult livers. On the other hand, an oligonucleotide probe specific to the coding region of NF25 and HLp (oli-NF) gave positive bands with RNA only from adult livers. These results indicate that P-450(HFL33) is expressed specifically in fetal livers and that neither P-450NF nor HLp is expressed in fetal livers, but one or both are expressed in adult livers.

Several organoselenium compounds including benzyl selenocyanate (BSC), 1,2-phenylenebis(methylene)selenocyanate (o-XSC), 1,3-phenylenebis(methylene)selenocyanate (m-XSC), and 1,4-phenylenebis(methylene)selenocyanate (p-XSC) have been shown to prevent cancers caused by polycyclic aromatic hydrocarbons (PAHs) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in experimental animals; these chemical carcinogens are activated by human P450 1 and 2A family enzymes, respectively, to carcinogenic metabolites. In this study, we examined whether these selenium compounds interact with and inhibit human P450 1 and 2A enzymes in vitro. Four organoselenium compounds induced Reverse Type I binding spectra with P450 1A1, 1A2, and 1B1 and Type I binding spectra with P450 2A6 and 2A13. The spectral dissociation constants (Ks) for the interaction of P450 1B1 with these chemicals were 3.6–5.7 µM; the values were lower than those with seen with P450 1A1 (19–30 µM) or 1A2 (6.3–13 µM). The Ks values for Type I binding of P450 2A13 with m-XSC and BSC were both 0.20 µM; the values were very low compared to the interaction of P450 2A6 with m-XSC (5.7 µM) and BSC (2.0 µM). Four selenium compounds directly inhibited 7-ethoxyresorufin O-deethylation activities catalyzed by P450 1A1, 1A2, and 1B1 with IC50 values <1.0 µM, except for the inhibition of P450 1A2 by BSC (1.3 µM). Coumarin 7-hydroxylation activities of P450 2A13 were more inhibited by four selenium compounds than those of P450 2A6, with IC50 values of 0.22–1.4 µM for P450 2A13 and 2.4–6.2 µM for P450 2A6. Molecular docking studies of the interaction of four organoselenium compounds with human P450 enzymes suggest that these chemicals can be docked into the active sites of these human P450 enzymes and that the sites of the selenocyanate functional groups of these chemicals differ between the P450 1 and 2A family enzymes.