Camptothecin bioprocessing from Aspergillus terreus, an endophyte of Catharanthus roseus: antiproliferative activity, topoisomerase inhibition and cell cycle analysis.

Attenuation of camptothecin (CPT) productivity by fungi with preservation and subculturing is the challenge that halts fungi to be an industrial platform of CPT production. Thus, screening for novel endophytic fungal isolates with metabolic stability for CPT production was the objective. Catharanthus roseus is one of the medicinal plants with diverse bioactive metabolites that could have a plethora of novel endophytes with unique metabolites. Among the endophytes of C. roseus, Aspergillus terreus EFBL-NV OR131583.1 had the most CPT producing potency (90.2 µg/l), the chemical identity of the putative CPT was verified by HPLC, FT-IR, NMR and LC-MS/MS. The putative A. terreus CPT had the same molecular mass (349 m/z), and molecular fragmentation patterns of the authentic one, as revealed from the MS/MS analyses. The purified CPT had a strong activity against MCF7 (5.27 µM) and UO-31 (2.2 µM), with a potential inhibition to Topo II (IC50 value 0.52 nM) than Topo 1 (IC50 value 6.9 nM). The CPT displayed a high wound healing activity to UO-31 cells, stopping their metastasis, matrix formation and cell immigration. The purified CPT had a potential inducing activity to the cellular apoptosis of UO-31 by ~ 17 folds, as well as, arresting their cellular division at the S-phase, compared to the control cells. Upon Plackett-Burman design, the yield of CPT by A. terreus was increased by ~ 2.6 folds, compared to control. The yield of CPT by A. terreus was sequentially suppressed with the fungal storage and subculturing, losing ~ 50% of their CPT productivity by 3rd month and 5th generation. However, the productivity of the attenuated A. terreus culture was completely restored by adding 1% surface sterilized leaves of C. roseus, and the CPT yield was increased over-the-first culture by ~ 3.2 folds (315.2 µg/l). The restoring of CPT productivity of A. terreus in response to indigenous microbiome of C. roseus, ensures the A. terreus-microbiome interactions, releasing a chemical signal that triggers the CPT productivity of A. terreus. This is the first reports exploring the potency of A. terreus, endophyte of C. roseus" to be a platform for industrial production of CPT, with an affordable sustainability with addition of C. roseus microbiome.

No Time to Relax and Unwind: Exploration of Topoisomerases and a Growing Field of Study.

With the topoisomerase field in its sixth decade [...].

Inborn errors of bile acid metabolism in Japan.

Bile acids are a category of steroids biosynthesized from cholesterol in the liver. Inborn errors of their metabolism are inherited in an autosomal recessive manner, resulting in enzyme deficiencies affecting the bile acid biosynthetic pathway. These defects in the pathway cause accumulation of unusual bile acids or bile alcohols. Unusual bile acids are highly cytotoxic, causing injury to the liver. These unusual bile acids damage hepatocytes, resulting in cholestatic liver injury beginning in infancy. Except for cerebrotendinous xanthomatosis and some secondary defects, various inborn errors of bile acid metabolism (IEBAM) have been reported from Japan, affecting eight patients including three with 3ß-hydroxy-Δ5 -C27 -steroid dehydrogenase/isomerase deficiency, three with Δ4 -3-oxosteroid 5ß-reductase deficiency, one with oxysterol 7α-hydroxylase deficiency, and one with bile acid-CoA: amino acid N-acyltransferase deficiency. Distinctive laboratory findings in patients with 3ß-hydroxy-Δ5 -C27 -steroid dehydrogenase/isomerase deficiency, Δ4 -3-oxosteroid 5ß-reductase deficiency, and oxysterol 7α-hydroxylase deficiency include normal serum γ-glutamyltransferase and total bile acids concentrations despite presence of cholestasis (elevated serum direct bilirubin) from infancy. Pediatricians and pediatric surgeons who suspect a case of IEBAM should obtain urinary and serum bile acid analyses using gas or liquid chromatography-mass spectrometry as well as genetic analyses. Available treatments include oral cholic acid, chenodeoxycholic acid, glycocholic acid, and ursodeoxycholic acid; fat-soluble vitamin supplementation; and liver transplantation. Early diagnosis and treatment can offer a good outcome.

Characterization of a new (Z)-3:(E)-2-hexenal isomerase from tea (Camellia sinensis) involved in the conversion of (Z)-3-hexenal to (E)-2-hexenal.

Two major green leaf volatiles (GLVs) in tea that contribute greatly to tea aroma, particularly the green odor, are (E)-2-hexenal and (Z)-3-hexenal. Until now, their formation and related mechanisms during tea manufacture have remained unclear. Our data showed that the contents of (E)-2-hexenal and (Z)-3-hexenal increased more than 1000-fold after live tea leaves were torn. Subsequently, a new (Z)-3:(E)-2-hexenal isomerase (CsHI) was identified in Camellia sinensis. CsHI irreversibly catalyzed the conversion of (Z)-3-hexenal to (E)-2-hexenal. Abiotic stresses including low temperature, dehydration, and mechanical wounding, did not influence the (E)-2-hexenal content in intact tea leaves during withering, but regulated the proportions of (Z)-3-hexenal and (E)-2-hexenal in torn leaves by modulating CsHI at the transcript level. For the first time, this work reveals the formation of (E)-2-hexenal during tea processing and suggests that CsHI may play a pivotal role in tea flavor development as well as in plant defense against abiotic stresses.

Genetic determinates for conjugated linolenic acid production in Lactobacillus plantarum ZS2058.

AIMS: To investigate the genetic determinates for conjugated linolenic acid (CLNA) production in Lactobacillus plantarum ZS2058, a high CLNA producer. METHODS AND RESULTS: After culturing with α-linolenic acid (ALA) in the medium, the fatty acid compositions of supernatant fluid and cell pellets were analysed via GC-MS. cis9,trans11,cis15-CLNA was identified to be the predominant isomer. And during CLNA production, 10-hydroxy-cis12-cis15-octadecenoic acid (10-HOEA) and 10-oxo-cis12-cis15-octadecenoic acid (10-OXOA) were accumulated. The E. coli recombinants harbouring genes encoding myosin-cross-reactive antigen (MCRA), short-chain dehydrogenase/oxidoreductase (DH) and acetoacetate decarboxylase (DC), respectively, were analysed for their roles in CLNA production. The results indicated that MCRA converted ALA to 10-HOEA, following converted to 10-OXOA by DH. While with the combination of three recombinants, ALA could be transformed into CLNA plus 10-HOEA and 10-OXOA. When the three genes were deleted, none of the L. plantarum ZS2058 knockout mutants could produce any CLNA, after complementation, and all the complementary mutants recovered the CLNA-production ability at similar levels as the wild strain. CONCLUSIONS: Lactobacillus plantarum ZS2058 produced CLNA from ALA with 10-HOEA and 10-OXOA as intermediates. The triple-component isomerase of MCRA, DH and DC was the unique genetic determinant for CLNA generation. SIGNIFICANCE AND IMPACT OF THE STUDY: The current results firstly provided conclusive evidence that the triple-component isomerase complex was shared by both CLA and CLNA production in lactobacilli.

Neopinone isomerase is involved in codeine and morphine biosynthesis in opium poppy.

The isomerization of neopinone to codeinone is a critical step in the biosynthesis of opiate alkaloids in opium poppy. Previously assumed to be spontaneous, the process is in fact catalyzed enzymatically by neopinone isomerase (NISO). Without NISO the primary metabolic products in the plant, in engineered microbes and in vitro are neopine and neomorphine, which are structural isomers of codeine and morphine, respectively. Inclusion of NISO in yeast strains engineered to convert thebaine to natural or semisynthetic opiates dramatically enhances formation of the desired products at the expense of neopine and neomorphine accumulation. Along with thebaine synthase, NISO is the second member of the pathogenesis-related 10 (PR10) protein family recently implicated in the enzymatic catalysis of a presumed spontaneous conversion in morphine biosynthesis.

Green Synthesis of Conjugated Linoleic Acids from Plant Oils Using a Novel Synergistic Catalytic System.

A novel and efficient method has been developed for converting plant oil into a specific conjugated linoleic acid (CLA) using a synergistic biocatalytic system based on immobilized Propionibacterium acnes isomerase (PAI) and Rhizopus oryzae lipase (ROL). PAI exhibited the greatest catalytic activity when immobilized on D301R anion-exchange resin under optimal conditions (PAI dosage of 12 410 U of PAI/g of D301R, glutaraldehyde concentration of 0.4%, and reaction conditions of pH 7.0, 25 °C, and 60 min). Up to 109 g/L trans-10,cis-12-CLA was obtained after incubation of 200 g/L sunflower oil with PAI (1659 U/g of oil) and ROL (625 mU/g of oil) at pH 7.0 and 35 °C for 36 h; the corresponding conversion ratio of linoleic acid (LA) to CLA was 90.5%. This method exhibited the highest proportion of trans-10,cis-12-CLA yet reported and is a promising method for large-scale production.

Selective Inhibition of Escherichia coli RNA and DNA Topoisomerase I by Hoechst 33258 Derived Mono- and Bisbenzimidazoles.

A series of Hoechst 33258 based mono- and bisbenzimidazoles have been synthesized and their Escherichia coli DNA topoisomerase I inhibition, binding to B-DNA duplex, and antibacterial activity has been evaluated. Bisbenzimidazoles with alkynyl side chains display excellent E. coli DNA topoisomerase I inhibition properties with IC50 values <5.0 µM. Several bisbenzimidazoles (3, 6, 7, 8) also inhibit RNA topoisomerase activity of E. coli DNA topoisomerase I. Bisbenzimidazoles inhibit bacterial growth much better than monobenzimidazoles for Gram-positive strains. The minimum inhibitory concentration (MIC) was much lower for Gram positive bacteria (Enterococcus spp. and Staphylococcus spp., including two MRSA strains 0.3-8 µg/mL) than for the majority of Gram negative bacteria (Pseudomonas aeruginosa, 16-32 µg/mL, Klebsiella pneumoniae > 32 µg/mL). Bisbenzimidazoles showed varied stabilization of B-DNA duplex (1.2-23.4 °C), and cytotoxicity studies show similar variation dependent upon the side chain length. Modeling studies suggest critical interactions between the inhibitor side chain and amino acids of the active site of DNA topoisomerase I.

Comprehensive identification and structural characterization of target components from Gelsemium elegans by high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry based on accurate mass databases combined with MS/MS spectra.

This study reports an applicable analytical strategy of comprehensive identification and structure characterization of target components from Gelsemium elegans by using high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QqTOF MS) based on the use of accurate mass databases combined with MS/MS spectra. The databases created included accurate masses and elemental compositions of 204 components from Gelsemium and their structural data. The accurate MS and MS/MS spectra were acquired through data-dependent auto MS/MS mode followed by an extraction of the potential compounds from the LC-QqTOF MS raw data of the sample. The same was matched using the databases to search for targeted components in the sample. The structures for detected components were tentatively characterized by manually interpreting the accurate MS/MS spectra for the first time. A total of 57 components have been successfully detected and structurally characterized from the crude extracts of G. elegans, but has failed to differentiate some isomers. This analytical strategy is generic and efficient, avoids isolation and purification procedures, enables a comprehensive structure characterization of target components of Gelsemium and would be widely applicable for complicated mixtures that are derived from Gelsemium preparations. Copyright © 2017 John Wiley & Sons, Ltd.

A metabolic engineering strategy for producing conjugated linoleic acids using the oleaginous yeast Yarrowia lipolytica.

Conjugated linoleic acids (CLAs) have been found to have beneficial effects on human health when used as dietary supplements. However, their availability is limited because pure, chemistry-based production is expensive, and biology-based fermentation methods can only create small quantities. In an effort to enhance microbial production of CLAs, four genetically modified strains of the oleaginous yeast Yarrowia lipolytica were generated. These mutants presented various genetic modifications, including the elimination of ß-oxidation (pox1-6∆), the inability to store lipids as triglycerides (dga1∆ dga2∆ are1∆ lro1∆), and the overexpression of the Y. lipolytica ∆12-desaturase gene (YlFAD2) under the control of the constitutive pTEF promoter. All strains received two copies of the pTEF-oPAI or pPOX-oPAI expression cassettes; PAI encodes linoleic acid isomerase in Propionibacterium acnes. The strains were cultured in neosynthesis or bioconversion medium in flasks or a bioreactor. The strain combining the three modifications mentioned above showed the best results: when it was grown in neosynthesis medium in a flask, CLAs represented 6.5% of total fatty acids and in bioconversion medium in a bioreactor, and CLA content reached 302 mg/L. In a previous study, a CLA degradation rate of 117 mg/L/h was observed in bioconversion medium. Here, by eliminating ß-oxidation, we achieved a much lower rate of 1.8 mg/L/h.

Branched-chain amino acid supplementation promotes aerobic growth of Salmonella Typhimurium under nitrosative stress conditions.

Nitric oxide (NO) inactivates iron-sulfur enzymes in bacterial amino acid biosynthetic pathways, causing amino acid auxotrophy. We demonstrate that exogenous supplementation with branched-chain amino acids (BCAA) can restore the NO resistance of hmp mutant Salmonella Typhimurium lacking principal NO-metabolizing enzyme flavohemoglobin, and of mutants further lacking iron-sulfur enzymes dihydroxy-acid dehydratase (IlvD) and isopropylmalate isomerase (LeuCD) that are essential for BCAA biosynthesis, in an oxygen-dependent manner. BCAA supplementation did not affect the NO consumption rate of S. Typhimurium, suggesting the BCAA-promoted NO resistance independent of NO metabolism. BCAA supplementation also induced intracellular survival of ilvD and leuCD mutants at wild-type levels inside RAW 264.7 macrophages that produce constant amounts of NO regardless of varied supplemental BCAA concentrations. Our results suggest that the NO-induced BCAA auxotrophy of Salmonella, due to inactivation of iron-sulfur enzymes for BCAA biosynthesis, could be rescued by bacterial taking up exogenous BCAA available in oxic environments.

Plant cyclopropylsterol-cycloisomerase: key amino acids affecting activity and substrate specificity.

The enzyme CPI (cyclopropylsterol-cycloisomerase) from the plant sterol pathway catalyses the cleavage of the 9ß,19-cyclopropane ring of the 4α-methyl-cyclopropylsterol cycloeucalenol to produce the Δ8-sterol obtusifoliol. Randomly mutated plasmids carrying the Arabidopsis thaliana cpi gene were screened for inactive CPI mutant enzymes on the basis of their ability to genetically complement a Saccharomyces cerevisiae erg7 (defective in oxidosqualene cyclase) ergosterol auxotroph grown in the presence of exogenous cycloeucalenol, and led to the identification of four catalytically important residues. Site-directed mutagenesis experiments confirmed the role of the identified residues, and demonstrated the importance of selected acidic residues and a conserved G108NYFWTHYFF117 motif. The mutated isomerases were assayed both in vivo by quantification of cycloeucalenol conversion into ergosterol in erg7 cells, and in vitro by examination of activities of recombinant AtCPI (A. thaliana CPI) mutants. These studies show that Gly28, Glu29, Gly108 and Asp260 are crucial for CPI activity and that an hydroxy function at residue 113 is needed for maximal substrate affinity and CPI activity. CPI is inactive on upstream 4α,ß-dimethyl-cyclopropylsterol precursors of phytosterols. The single mutation W112L generates a CPI with an extended substrate specificity, that is able to convert 4α,ß-dimethyl-cyclopropylsterols into the corresponding Δ8 products. These findings provide insights into the molecular basis of CPI activity and substrate specificity.

Production of taxadiene from cultured ginseng roots transformed with taxadiene synthase gene.

Paclitaxel is produced by various species of yew trees and has been extensively used to treat tumors. In our research, a taxadiene synthase (TS) gene from Taxus brevifolia was used to transform the roots of cultured ginseng (Panax ginseng C.A. Meyer) to produce taxadiene, the unique skeletal precursor to taxol. The TS gene was successfully introduced into the ginseng genome, and the de novo formation of taxadiene was identified by mass spectroscopy profiling. Without any change in phenotypes or growth difference in a TS-transgenic ginseng line, the transgenic TSS3-2 line accumulated 9.1 µg taxadiene per gram of dry weight. In response to the treatment of methyl jasmonate for 3 or 6 days, the accumulation was 14.6 and 15.9 µg per g of dry weight, respectively. This is the first report of the production of taxadiene by engineering ginseng roots with a taxadiene synthase gene.

NdgR, a common transcriptional activator for methionine and leucine biosynthesis in Streptomyces coelicolor.

We show here that NdgR, a known transcriptional activator of isopropylmalate dehydratase in actinomycetes, may have other targets in the cell. An in-frame deletion mutant of ndgR showed unexpectedly poor growth in defined minimal medium even in the presence of leucine. To our surprise, it was supplementation of cysteine and methionine that corrected the growth. Based on this, we propose that NdgR induces cysteine-methionine biosynthesis. Direct involvement of NdgR in the very last steps of methionine synthesis with methionine synthase (metH) and 5,10-methylenetetrahydrofolate reductase (metF) was examined. From a pulldown assay, it was seen that NdgR was enriched from crude cell lysates with a strong affinity to metH and metF upstream sequences. Direct physical interaction of NdgR with these targets was further examined with a gel mobility shift assay. ndgR, leuC, metH, and metF were inducible in M145 cells upon nutrient downshift from rich to minimal medium but were not induced in the ndgR knockout mutant. Taking these observations together, NdgR-dependent metH-metF expression would account for the abnormal growth phenotype of the ndgR mutant although there may be additional NdgR-dependent genes in the Cys-Met metabolic pathways. As the first transcriptional factor reported for regulating Cys-Met metabolism in Streptomyces, NdgR links two disparate amino acid families, branched-chain amino acids (BCAAs) and sulfur amino acids, at the transcriptional level. Considering that Cys-Met metabolism is connected to mycothiol and one-carbon metabolism, NdgR may have broad physiological impacts.

Toward pectin fermentation by Saccharomyces cerevisiae: expression of the first two steps of a bacterial pathway for D-galacturonate metabolism.

Saccharomyces cerevisiae cannot metabolize D-galacturonate, an important monomer of pectin. Use of S. cerevisiae for production of ethanol or other compounds of interest from pectin-rich feedstocks therefore requires introduction of a heterologous pathway for D-galacturonate metabolism. Bacterial D-galacturonate pathways involve D-galacturonate isomerase, D-tagaturonate reductase and three additional enzymes. This study focuses on functional expression of bacterial D-galacturonate isomerases in S. cerevisiae. After demonstrating high-level functional expression of a D-tagaturonate reductase gene (uxaB from Lactococcus lactis), the resulting yeast strain was used to screen for functional expression of six codon-optimized bacterial D-galacturonate isomerase (uxaC) genes. The L. lactis uxaC gene stood out, yielding a tenfold higher enzyme activity than the other uxaC genes. Efficient expression of D-galacturonate isomerase and D-tagaturonate reductase represents an important step toward metabolic engineering of S. cerevisiae for bioethanol production from D-galacturonate. To investigate in vivo activity of the first steps of the D-galacturonate pathway, the L. lactis uxaB and uxaC genes were expressed in a gpd1Δ gpd2Δ S. cerevisiae strain. Although D-tagaturonate reductase could, in principle, provide an alternative means for re-oxidizing cytosolic NADH, addition of D-galacturonate did not restore anaerobic growth, possibly due to absence of a functional D-altronate exporter in S. cerevisiae.

Detection of platypus-type L/D-peptide isomerase activity in aqueous extracts of papaya fruit.

Peptide isomerase catalyses the post-translational isomerisation of the L: - to the D: -form of an amino acid residue around the N/C-termini of substrate peptides. To date, some peptide isomerases have been found in a limited number of animal secretions and cells. We show here that papaya extracts have weak peptide isomerase activity. The activity was detected in each 30-100 kDa fraction of the flesh and the seed extracts of unripe and ripe papaya fruit. The definitive activity was confirmed in the ripe papaya extracts, but even then it was much less active than that of the other peptide isomerases previously reported. The activity was markedly inhibited by methanol, and partly so by amastatin and diethyl pyrocarbonate. This is the first report of peptide isomerase activity in a plant and suggests that perhaps every living organism may have some peptide isomerase activity.

Titration of bile acid supplements in 3beta-hydroxy-Delta 5-C27-steroid dehydrogenase/isomerase deficiency.

OBJECTIVES: 3beta-Hydroxy-Delta 5-C27-steroid dehydrogenase/isomerase deficiency is a bile acid synthesis defect responsive to primary bile acids. We reviewed its clinical features and response to treatment with a mixture of ursodeoxycholic (UDCA) and chenodeoxycholic acid (CDCA) to titrate the dose of supplements required for disease control. PATIENTS AND METHODS: We studied our patients by liquid chromatography-tandem mass spectrometry, liver function tests, and histology. After diagnosis all of the patients received a balanced mixture of UDCA/CDCA and the dose was titrated according to urinary levels of 3beta,7 alpha-dihydroxy-5-cholenoic acid (u-3beta-D-OH-5C). RESULTS: Five patients presenting with giant cell hepatitis, biliary cirrhosis, and cryptogenic cirrhosis (1 each), and picked up by neonatal screening (2 patients) were diagnosed at a median age of 2.5 years (range 0.1-5.5). Normal levels of u-3beta-D-OH-5C were achieved after 4 months (range 3-28 months) from the start of the treatment. The minimum dose of UDCA/CDCA required to maintain normal u-3beta-D-OH-5C levels was 5/5 mg x kg(-1) x day(-1). A follow-up biopsy in 2 patients showed no progression of liver disease. CONCLUSIONS: A mixture of UDCA/CDCA can effectively control 3beta-hydroxy-Delta 5-C27-steroid dehydrogenase/isomerase deficiency. Dose titration by liquid chromatography-tandem mass spectrometry warrants the maintenance of negative feedback on the abnormal synthetic pathway and avoids disease progression.

Taxomyces andreanae: a presumed paclitaxel producer demystified?

The 1990s brought an abundance of reports on paclitaxel-producing endophytes, initially heralded as a discovery having tremendous implications for cancer therapy. As the vision of large-scale fermentation tanks producing vast quantities of relatively inexpensive paclitaxel and novel taxanes has faded and has been replaced by controversial silence, we carried out an in-depth investigation of Taxomyces andreanae - the very first presumed endophytic synthesizer of the diterpenoid. On one hand, metabolic profiling by means of chromatographic, spectroscopic and immunoenzymatic techniques predominant in literature was taken up. On the other, the experimental procedure was brought to an alternative, previously unattempted level aiming at revealing the genetic background of paclitaxel biosynthesis in the endophyte. The profound PCR-based screening for taxadiene synthase (TXS) - a gene unique to the formation of the primary taxane-skeleton, as well as phenylpropanoyl transferase (BAPT) encoding for the catalyst of the final acylation of the core structure rendering the ultimate efficacy of the drug, confirmed the molecular blueprint for paclitaxel biosynthesis to be an inherent genetic trait of the endophyte. However, as the thorough metabolic analysis of Taxomyces andreanae commercial isolate brought no confirmation of endophytic paclitaxel production even after considerable up-scaling endeavors, we postulate that proclaiming the strain "a fungus factory for Taxol" might have been premature.

Analysis of Amaryllidaceae alkaloids from Crinum by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry.

A high-performance liquid chromatography/electrospray ionization multi-stage tandem mass spectrometry (HPLC/ESI-MS(n)) method was developed to analyze two structurally related groups of Amaryllidaceae alkaloids (AmAs), crinane- and tazettine-type alkaloids, in the species Crinum latifolium and C. asiaticum, as well as different organs of C. latifolium. In ESI-MS(n) spectra of the two types of alkaloids, characteristic fragmentation reactions were observed that allowed us to determine and differentiate them. Based on the fragmentation rules of reference standards, crinane-type alkaloids displayed concurrent neutral loss of C(2)H(5)N (43 u) and C(2)H(6)N (44 u) as well as characteristic ions of m/z 213 and 211, whereas tazettine-type alkaloids exhibited neutral loss of C(3)H(7)N (57 u) [or C(2)H(5)N (43 u), C(3)H(7)NO (73 u)] from the [M+H](+) and [M+H-H(2)O](+) ions. These were supported by quadrupole time-of-flight (Q-Tof)-MS/MS analysis. The chemical complexity of the mixture was resolved by profiling. The compositions of the main crinane- and tazettine-type alkaloids in the above-mentioned species and organs were also compared. Overall, 28 AmAs comprising 14 crinane-type and 14 tazettine-type alkaloids were identified and studied by MS. Among them, 14 AmAs were tentatively characterized from the two species for the first time. This method allowed a rapid analysis of alkaloid distribution and composition of Crinum species, and may also be used for quality control and screening of extracts designated for pharmaceutical application.

The pesticide adjuvant, Toximul, alters hepatic metabolism through effects on downstream targets of PPARalpha.

Previous studies demonstrated that chronic dermal exposure to the pesticide adjuvant (surfactant), Toximul (Tox), has significant detrimental effects on hepatic lipid metabolism. This study demonstrated that young mice dermally exposed to Tox for 12 days have significant increases in expression of peroxisomal acyl-CoA oxidase (mRNA and protein), bifunctional enzyme (mRNA) and thiolase (mRNA), as well as the P450 oxidizing enzymes Cyp4A10 and Cyp4A14 (mRNA and protein). Tox produced a similar pattern of increases in wild type adult female mice but did not induce these responses in PPARalpha-null mice. These data support the hypothesis that Tox, a heterogeneous blend of nonionic and anionic surfactants, modulates hepatic metabolism at least in part through activation of PPARalpha. Notably, all three groups of Tox-treated mice had increased relative liver weights due to significant accumulation of lipid. This could be endogenous in nature and/or a component(s) of Tox or a metabolite thereof. The ability of Tox and other hydrocarbon pollutants to induce fatty liver despite being PPARalpha agonists indicates a novel consequence of exposure to this class of chemicals, and may provide a new understanding of fatty liver in populations with industrial exposure.