Cytotoxic and Membrane Cholesterol Effects of Ultraviolet Irradiation and Zinc Oxide Nanoparticles on Chinese Hamster Ovary Cells.

Zinc Oxide (ZnO) nanoparticles are suspected to produce toxic effects toward mammalian cells; however, discrepancies in the extent of this effect have been reported between different cell lines. Simultaneously, high levels of ultraviolet (UV-C) radiation can have carcinogenic effects. The mechanism of this effect is also not well understood. Due to similarities in phenotype morphology after cell exposure to ZnO nanoparticles and UV-C irradiation, we emit the hypothesis that the toxicity of both these factors is related to damage of cellular membranes and affect their sterol content. Wild-type Chinese Hamster Ovary (CHO-K1) cells were exposed to ZnO nanoparticles or UV-C radiation. The amount of absorbed ZnO was determined by UV-visible spectroscopy and the changes in sterol profiles were evaluated by gas chromatography. Cell viability after both treatments was determined by microscopy. Comparing morphology results suggested similarities in toxicology events induced by ZnO nanoparticles and UV exposure. UV-C exposure for 360 min disrupts the sterol metabolic pathway by increasing the concentration of cholesterol by 21.6-fold. This increase in cholesterol production supports the hypothesis that UV irradiation has direct consequences in initiating sterol modifications in the cell membrane.

Purification, characterization and inhibition of sterol C24-methyltransferase from Candida albicans.

Solubilized sterol C24-methyltransferase (24-SMT) was purified to homogeneity from a cell extract of the yeast Candida albicans (Ca) by anion exchange chromatography, gel permeation chromatography and fast performance liquid chromatography using a Mono Q column. The purified enzyme has an apparent molecular mass of 178 kDa on gel permeation chromatography and 43 kDa on SDS/PAGE, indicating that it is composed of four identical subunits. The substrate requirement of the native enzyme has an optimal specificity for zymosterol with associated kinetic constants of K(m) 50 µM and k(cat) of 0.01 s⁻¹. The product of the enzyme incubated with zymosterol was fecosterol. Inhibition of the catalyst was observed with substrate analogs designed as transition state analogs (25-azalanosterol, K(i)=54 nM and 24 (R,S),25-epiminolanosterol, K(i)=11 nM) or as mechanism-based inactivators (26,27-dehydrozymosterol, K(i) 9 µM) and k(inact)=0.03 min⁻¹) of the C24-methylation reaction. Product analogs ergosterol and fecosterol, but neither cholesterol nor sitosterol, inhibited activity affording K(i) values of 20 and 72 µM, respectively. Ammonium and thia analogs of the intermediates of the sterol C24-methyl reaction sequence were effective growth inhibitors exhibiting IC(50) values that ranged from 3 to 20 µM.

Cloning, mechanistic and functional analysis of a fungal sterol C24-methyltransferase implicated in brassicasterol biosynthesis.

The first committed step in the formation of 24-alkylsterols in the ascomycetous fungus Paracoccidiodes brasiliensis (Pb) has been shown to involve C24-methylation of lanosterol to eburicol (24(28)-methylene-24,25-dihydro-lanosterol) on the basis of metabolite co-occurrence. A similarity-based cloning strategy was employed to obtain the cDNA clone corresponding to the sterol C24-methyltransferase (SMT) implicated in the C24-methylation reaction. The resulting catalyst, prepared as a recombinant fusion protein (His/Trx/S), was expressed in Escherichia coli BL21(C43) and shown to possess a substrate specificity for lanosterol and to generate a single exocyclic methylene product. The full-length cDNA has an open reading frame of 1131 base pairs and encodes a protein of 377 residues with a calculated molecular mass of 42,502Da. The enzymatic C24-methylation gave a K(mapp) of 38microM and k(catapp) of 0.14min(-1). Quite unexpectedly, "plant" cycloartenol was catalyzed in high yield to 24(28)-methylene cycloartanol consistent with conformational arguments that favor that both cycloartenol and lanosterol are bound pseudoplanar in the ternary complex. Incubation of [27-(13)C]- or [24-(2)H]cycloartenol with PbSMT and analysis of the enzyme-generated product by a combination of (1)H and (13)CNMR and mass spectroscopy established the regiospecific conversion of the pro-Z methyl group of the Delta(24(25))-substrate to the pro-R isopropyl methyl group of the product and the migration of H24 to C25 on the Re-face of the original substrate double bond undergoing C24-methylation. Inhibition kinetics and products formed from the substrate analogs 25-azalanosterol (K(i) 14nM) and 26,27-dehydrolanosterol (K(i) 54muM and k(inact) of 0.24min(-1)) provide direct evidence for distinct reaction channeling capitalized by structural differences in the C24- and C26-sterol acceptors. 25-Azalanosterol was a potent inhibitor of cell growth (IC(50), 30nM) promoting lanosterol accumulation and 24-alkyl sterol depletion. Phylogenetic analysis of PbSMT with related SMTs of diverse origin together with the results of the present study indicate that the enzyme may have a similar complement of active-site amino acid residues compared to related yeast SMTs affording monofunctional C(1)-transfer behavior, yet there are sufficient differences in its overall amino acid composition and substrate-dependent partitioning pathways to group PbSMT into a fourth and new class of SMT.

Disruption of ergosterol biosynthesis, growth, and the morphological transition in Candida albicans by sterol methyltransferase inhibitors containing sulfur at C-25 in the sterol side chain.

The sterol substrate analog 25-thialanosterol and its corresponding sulfonium salt were evaluated for their ability to serve as antifungal agents and to inhibit sterol methyltransferase (SMT) activity in Candida albicans. Both compounds inhibited cell proliferation, were fungistatic, interrupted the yeast-like-form to germ-tube-form transition, and resulted in the accumulation of zymosterol and related delta24-sterols concurrent with a decrease in ergosterol, as was expected for the specific inhibition of SMT activity. Feedback on sterol synthesis was evidenced by elevated levels of cellular sterols in treated vs. control cultures. However, neither farnesol nor squalene accumulated in significant amounts in treated cultures, suggesting that carbon flux is channeled from the isoprenoid pathway to the sterol pathway with minor interruption or redirection until blockage at the C-methylation step. Activity assays using solubilized C. albicans SMT confirmed the inhibitors impair SMT action. Kinetic analysis indicated that 25-thialanosterol inhibited SMT with the properties of a time-dependent mechanism-based inactivator Ki of 5 microM and apparent kinact of 0.013 min(-1), whereas the corresponding sulfonium salt was a reversible-type transition state analog exhibiting a Ki of 20 nM. The results are interpreted to imply changes in ergosterol homeostasis as influenced by SMT activity can control growth and the morphological transition in C. albicans, possibly affecting disease development.

Evidence for multiple sterol methyl transferase pathways in Pneumocystis carinii.

The sterol composition of Pneumocystis carinii, an opportunistic pathogen responsible for life-threatening pneumonia in immunocompromised patients, was determined. Our purpose was to identify pathway-specific enzymes to impair using sterol biosynthesis inhibitors. Prior to this study, cholesterol 15 (ca. 80% of total sterols), lanosterol 1, and several phytosterols common to plants (sitosterol 31, 24alpha-ethyl and campesterol, 24alpha-methyl 30) were demonstrated in the fungus. In this investigation, we isolated all the previous sterols and many new compounds from P. carinii by culturing the microorganism in steroid-immunosuppressed rats. Thirty-one sterols were identified from the fungus (total sterol = 100 fg/cell), and seven sterols were identified from rat chow. Unusual sterols in the fungus not present in the diet included, 24(28)-methylenelanosterol 2; 24(28)E-ethylidene lanosterol 3; 24(28)Z-ethylidene lanosterol 4; 24beta-ethyllanosta-25(27)-dienol 5; 24beta-ethylcholest-7-enol 6; 24beta-ethylcholesterol 7; 24beta,-ethylcholesta-5,25(27)-dienol 8; 24-methyllanosta-7-enol 9; 24-methyldesmosterol 10; 24(28)-methylenecholest-7-enol 11; 24beta-methylcholest-7-enol 12; and 24beta-methylcholesterol 13. The structural relationships of the 24-alkyl groups in the sterol side chain were demonstrated chromatographically relative to authentic specimens, by MS and high-resolution 1H NMR. The hypothetical order of these compounds poses multiple phytosterol pathways that diverge from a common intermediate to generate 24beta-methyl sterols: route 1, 1 --> 2 --> 11 --> 12 --> 13; route 2, 1 --> 2 --> 9 --> 10 --> 13; or 24beta-ethyl sterols: route 3, 1 --> 2 --> 4 --> 6 --> 7; route 4, 1 --> 2 --> 5 --> 8 --> 7. Formation of 3 is considered to form an interrupted sterol pathway. Taken together, operation of distinct sterol methyl transferase (SMT) pathways that generate 24beta-alkyl sterols in P. carinii with no counterpart in human biochemistry suggests a close taxonomic affinity with fungi and provides a basis for mechanism-based inactivation of SMT enzyme to treat Pneumocystis pneumonia.