Effects of Deoxynivalenol and Fumonisins Fed in Combination to Beef Cattle: Immunotoxicity and Gene Expression.

We evaluated the effects of a treatment diet contaminated with 1.7 mg deoxynivalenol and 3.5 mg fumonisins (B1, B2 and B3) per kg ration on immune status and peripheral blood gene expression profiles in finishing-stage Angus steers. The mycotoxin treatment diet was fed for a period of 21 days followed by a two-week washout period during which time all animals consumed the control diet. Whole-blood leukocyte differentials were performed weekly throughout the experimental and washout period. Comparative profiles of CD4+ and CD8+ T cells, along with bactericidal capacity of circulating neutrophils and monocytes were evaluated at 0, 7, 14, 21 and 35 days. Peripheral blood gene expression was measured at 0, 7, 21 and 35 days via RNA sequencing. Significant increases in the percentage of CD4-CD8+ T cells were observed in treatment-fed steers after two weeks of treatment and were associated with decreased CD4:CD8 T-cell ratios at this same timepoint (p ≤ 0.10). No significant differences were observed as an effect of treatment in terms of bactericidal capacity at any timepoint. Dietary treatments induced major changes in transcripts associated with endocrine, metabolic and infectious diseases; protein digestion and absorption; and environmental information processing (inhibition of signaling and processing), as evaluated by dynamic impact analysis. DAVID analysis also suggested treatment effects on oxygen transport, extra-cellular signaling, cell membrane structure and immune system function. These results indicate that finishing-stage beef cattle are susceptible to the immunotoxic and transcript-inhibitory effects of deoxynivalenol and fumonisins at levels which may be realistically encountered in feedlot situations.

Molecular and Alkaloid Characterization of Claviceps purpurea Sensu Lato From Grass Seed Production Areas of the U.S. Pacific Northwest.

Ergot, caused by Claviceps purpurea sensu lato, is an economically important seed replacement disease of Kentucky bluegrass (Poa pratensis) and perennial ryegrass (Lolium perenne) seed crops. C. purpurea sensu stricto is considered the primary Claviceps species responsible, but genetic diversity and cryptic species within C. purpurea sensu lato have previously been reported. Fifty-six C. purpurea sensu lato isolates collected from P. pratensis (n = 21) and L. perenne (n = 35) in Oregon and Washington between 2010 and 2014 were characterized via random amplified polymorphic DNA (RAPD), partial internal transcribed spacer (ITS), ß-tubulin and elongation factor-1α (EF-1α) sequences, conidial size, and ergot alkaloid chemotype. Based on RAPD analysis, seven isolates from P. pratensis and 33 isolates from L. perenne collected in Oregon corresponded to C. purpurea sensu stricto, and 13 isolates collected from P. pratensis in Washington and Oregon were identified as C. humidiphila. Partial ITS, ß-tubulin, and EF-1α sequences identified 10 isolates from P. pratensis as C. humidiphila, and seven isolates from P. pratensis and 33 isolates from L. perenne were identified as C. purpurea sensu stricto. Several isolates generated ambiguous RAPD bands or sequences that prevented identification. Ergot alkaloid chemotype profiling found that ergocornine and its epimer were predominant in sclerotia from P. pratensis, whereas ergotamine and its epimer were most abundant in sclerotia from L. perenne. This study confirms the presence of the C. purpurea sensu lato species complex in the U.S. Pacific Northwest and suggests that more research is needed to characterize and mitigate Claviceps spp. infection of grass seed crops in North America.

Mortality of Potato Psyllid (Hemiptera: Triozidae) on Host Clippings Inoculated With Ergot Alkaloids.

Our previous study provided correlative evidence that morning glory species harboring endophytic fungi (Periglandula) are resistant to potato psyllid [Bactericera cockerelli (Sulc)], whereas species free of fungi often allowed psyllid development. In this study, we manipulated levels of ergot alkaloids in host tissues by inoculating clippings from potato plants with extracts from morning glories that harbor Periglandula [Ipomoea leptophylla Torrey, Ipomoea imperati (Vahl) Grisebach, Ipomoea tricolor Cavanilles, Ipomoea pandurata (L.) G. F. Meyer, and Turbina corymbosa (L.)] and one species (Ipomoea alba L.) that does not harbor the endophyte. Ergot alkaloids (clavines, lysergic acid amides, and ergopeptines) were detected in potato clippings, thus confirming that leaves had taken up compounds from solutions of crude extracts. Psyllid mortality rates on inoculated clippings ranged between 53 and 93% in treatments producing biochemically detectable levels of alkaloids, when compared with 15% mortality in water controls or the alkaloid-free I. alba. We then tested synthetic analogs from each of the three alkaloid classes that had been detected in the crude extracts. Each compound was assayed by inoculating clippings of two host species (potato and tomato) at increasing concentrations (0, 1, 10, and 100 µg/ml in solution). Psyllids exhibited a large and significant increase in mortality rate beginning at the lowest two concentrations, indicating that even very small quantities of these chemicals led to mortality. Feeding by nymphs on artificial diets containing synthetic compounds resulted in 100% mortality within 48 h, irrespective of compound. Further testing of ergot alkaloids to characterize the mode of action that leads to psyllid mortality is warranted.

Survival and development of potato psyllid (Hemiptera: Triozidae) on Convolvulaceae: Effects of a plant-fungus symbiosis (Periglandula).

Plant species in the family Solanaceae are the usual hosts of potato psyllid, Bactericera cockerelli (Sulc) (Hemiptera: Psylloidea: Triozidae). However, the psyllid has also been shown to develop on some species of Convolvulaceae (bindweeds and morning glories). Developmental success on Convolvulaceae is surprising given the rarity of psyllid species worldwide associated with this plant family. We assayed 14 species of Convolvulaceae across four genera (Convolvulus, Calystegia, Ipomoea, Turbina) to identify species that allow development of potato psyllid. Two populations of psyllids were assayed (Texas, Washington). The Texas population overlaps extensively with native Convolvulaceae, whereas Washington State is noticeably lacking in Convolvulaceae. Results of assays were overlain on a phylogenetic analysis of plant species to examine whether Convolvulaceae distantly related to the typical host (potato) were less likely to allow development than species of Convolvulaceae more closely related. Survival was independent of psyllid population and location of the plant species on our phylogenetic tree. We then examined whether presence of a fungal symbiont of Convolvulaceae (Periglandula spp.) affected psyllid survival. These fungi associate with Convolvulaceae and produce a class of mycotoxins (ergot alkaloids) that may confer protection against plant-feeding arthropods. Periglandula was found in 11 of our 14 species, including in two genera (Convolvulus, Calystegia) not previously known to host the symbiont. Of these 11 species, leaf tissues from five contained large quantities of two classes of ergot alkaloids (clavines, amides of lysergic acid) when evaluated by LC-MS/MS. All five species also harbored Periglandula. No ergot alkaloids were detected in species free of the fungal symbiont. Potato psyllid rapidly died on the five species that harbored Periglandula and contained ergot alkaloids, but survived to adulthood on seven of the nine species in which ergot alkaloids were not detected. These results support the hypothesis that a plant-fungus symbiotic relationship affects the suitability of certain Convolvulaceae to potato psyllid.

Co-exposure of the Mycotoxins Lolitrem B and Ergovaline in Steers Fed Perennial Ryegrass ( Lolium perenne) Straw: Metabolic Characterization of Excreta.

Past research showed a strong linear correlation between levels of the mycotoxins lolitrem B (LB, a tremorgen) and ergovaline (EV, an ergot alkaloid and potent vasoconstrictor) in perennial ryegrass (PRG) forage. The purpose of this study was to characterize the excretion of these two compounds in beef cattle consuming PRG straw and to utilize liquid chromatography-tandem mass spectrometry to investigate the metabolism of LB and EV in excreta. Four groups of steers ( n = 6/group) were fed endophyte-infected PRG for 64 days (2256/638, 1554/373, 1012/259, or 247/<100 µg/kg LB/EV). Concentrations of LB and EV in both PRG straw and feces showed a linear relationship to each other. Feces reflected a dose-response for both mycotoxins, with values increasing most rapidly through 21 days then plateauing. Urine contained no detectable level of either compound or the ergoline lysergic acid. Screening for metabolites showed oxidation and reduction biotransformations for both toxins, with additional conjugation products detected for ergovaline.

Volatile Metabolomic Composition of Vitex Species: Chemodiversity Insights and Acaricidal Activity.

The Vitex genus (Lamiaceae) produces a plethora of metabolites that include ecdysteroids and terpenoids, some of which have demonstrated insect repellent properties. The volatile composition of several members of this genus has not been chemically defined, as many taxa are endemic to remote ecosystems. In this study, leaves were collected from the northeast of Brazil from Vitex capitata, V. megapotamica, V. gardneriana, and V. rufescens plants and examined for their chemical profile via GC-MS/FID of essential oil extracts. The analyses showed a diversity of terpenoids. Of particular note were seven-member ring sesquiterpenes which were present in great abundance; a dendrogram showed clades separating by the production of bicyclogermacrene, aromadendrane and 5,10-cycloaromadendrane sesquiterpenoids for the four species. Comparison of volatile metabolite profiles to 13 other Vitex species showed strong similarities in the production of some monoterpenes, but varied by their production of larger terpenes, especially those with gem-dimethylcyclopropyl subunits on seven-member ring compounds. From this work, we suggest that the sesquiterpene skeleton with seven member rings is a good chemosystematic biomarker candidate for the Vitex genus. Separation using this biomarker was then validated using Inter-Simple Sequence Repeat profiling. Lastly, experiments examining the toxicity of these four oils against the coconut mite Aceria guerreronis showed that only the oil of V. gardneriana had strong acaricidal activity, with an LC50 of 0.85 mg/mL, thus demonstrating its potential for use as a natural pesticide.

Cases of ergotism in livestock and associated ergot alkaloid concentrations in feed.

Ergot-induced disease in humans was known long before Biblical times and has been the root cause for countless human epidemics spanning from the early fourteenth century to the late sixteenth century. In contrast, many of these same ergot alkaloids have been utilized for their medicinal properties to mitigate migraine headaches and have had indications as anti-carcinogens. Although ergot alkaloids have been used for centuries by humans, basic pharmacokinetic data has not been documented for clinical disease in livestock. Consequently, a threshold dose and accurate dose-response data have yet to be established. Throughout the past several years, new detection techniques have emerged to detect these alkaloids at the parts per billion (ppb) level which has allowed for new efforts to be made with respect to determining threshold levels and making accurate clinical diagnoses in affected animals. This perspectives article provides a critical initial step for establishing a uniform interpretation of ergot toxicosis from limited existing data.

Metagenomic insights into the RDX-degrading potential of the ovine rumen microbiome.

The manufacturing processes of royal demolition explosive (RDX), or hexahydro-1,3,5-trinitro-1,3,5-triazine, have resulted in serious water contamination. As a potential carcinogen, RDX can cause a broad range of harmful effects to humans and animals. The ovine rumen is capable of rapid degradation of nitroaromatic compounds, including RDX. While ruminal RDX-degrading bacteria have been identified, the genes and pathways responsible for RDX degradation in the rumen have yet to be characterized. In this study, we characterized the metabolic potential of the ovine rumen using metagenomic approaches. Sequences homologous to at least five RDX-degrading genes cloned from environmental samples (diaA, xenA, xenB, xplA, and xplB) were present in the ovine rumen microbiome. Among them, diaA was the most abundant, likely reflective of the predominance of the genus Clostridium in the ovine rumen. At least ten genera known to harbor RDX-degrading microorganisms were detectable. Metagenomic sequences were also annotated using public databases, such as Pfam, COG, and KEGG. Five of the six Pfam protein families known to be responsible for RDX degradation in environmental samples were identified in the ovine rumen. However, increased substrate availability did not appear to enhance the proliferation of RDX-degrading bacteria and alter the microbial composition of the ovine rumen. This implies that the RDX-degrading capacity of the ovine rumen microbiome is likely regulated at the transcription level. Our results provide metagenomic insights into the RDX-degrading potential of the ovine rumen, and they will facilitate the development of novel and economic bioremediation strategies.

The role of the Oregon State University Endophyte Service Laboratory in diagnosing clinical cases of endophyte toxicoses.

The Oregon State University Colleges of Veterinary Medicine and Agricultural Sciences instituted the Endophyte Service Laboratory to aid in diagnosing toxicity problems associated with cool-season grasses in livestock. The endophyte (Neotyphodium coenophalum) present in tall fescue (Festuca arundinacea) produces ergopeptine alkaloids, of which ergovaline is the molecule used to determine exposure and toxicity thresholds for the vasoconstrictive conditions "fescue foot" and "summer slump". Another vasoconstrictive syndrome, "ergotism," is caused by a parasitic fungus, Claviceps purpurea, and its primary toxin, ergotamine. "Ryegrass staggers" is a neurological condition that affects livestock consuming endophyte (Neotyphodium lolii)-infected perennial ryegrass (Lolium perenne) with high levels of lolitrem B. HPLC-fluorescent analytical methods for these mycotoxins are described and were used to determine threshold levels of toxicity for ergovaline and lolitrem B in cattle, sheep, horses, and camels. In addition, six clinical cases in cattle are presented to illustrate diagnosis of these three diseases.

Ruminal bioremediation of the high energy melting explosive (HMX) by sheep microorganisms.

The ability of ruminal microorganisms to degrade octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (high melting explosive, HMX) as consortia from whole rumen fluid (WRF), and individually as 23 commercially available ruminal strains, was compared under anaerobic conditions. Compound degradation was monitored by high-performance liquid chromatography, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) for delineation of the metabolic pathway. In WRF, 30 µM HMX was degraded to 5 µM HMX within 24 h. Metabolites consistent with m/z 149, 193 and 229 were present throughout the incubation period. We propose that peaks with an m/z of 149 and 193 are arrived at through reduction of HMX to nitroso or hydroxylamino intermediates, then direct enzymatic ring cleavage to produce these HMX derivatives. Possible structures of m/z 229 are still being investigated and require further LC-MS/MS analysis. None of the 23 ruminal strains tested were able to degrade HMX as a pure culture when grown in either a low carbon or low nitrogen basal medium over 120 h. We conclude that microorganisms from the rumen, while sometimes capable as individuals in the bioremediation of other explosives, excel as a community in the case of HMX breakdown.

ESI-Mass spectrometric and HPLC elucidation of a new ergot alkaloid from perennial ryegrass hay silage associated with bovine reproductive problems.

This case report involves four dairies in the Willamette Valley, Oregon, which experienced reproductive problems associated with the presence of a large, previously unidentified, peak eluting at 5 min in a standard ergovaline high-performance liquid chromatography assay of perennial ryegrass silage fed to those animals. Mycotoxin analysis of the silage was negative, as was serological screening of the herds for infectious bovine rhinotracheitis, bovine diarrhea virus and Leptospirosis, including culturing of urine for Leptospira hardjo hardjobovis. Prolactin concentrations were low in most cattle, consistent with ingestion of ergot alkaloids. We believe that this peak represents a novel ergot alkaloid-related compound due to its extractability with Ergosil, its detectability due to fluorescence, and its chromatographic retention between ergovaline (mw = 533) and ergotamine (mw = 581). Its molecular weight was calculated as 570 owing to the predominance of a m/z 593.5 ion in the full scan ESI(+)MS and its deduced tendency to complex with Na(+) (as m/z 593) or K(+) (as m/z 609) ions. We offer rationales for elucidation of the structure of this compound, with the closest starting point comprising an m.w. of 566-a fructofuranosyl-(2-1)-O-beta-D-fructofuranoside derivative of 6,7-secoergoline from Claviceps fusiformis. This m.w. requires modifications, such as reduction of two double bonds in the secoergoline component to give the target 570 m.w. Despite the lack of a definitive structure, the analysis herein provides a starting point for eventual elucidation of this apparently new ergot alkaloid, and to guide and encourage further investigation as to its association with endophyte toxicosis in livestock.

Composition and antibacterial activity of essential oils obtained from plants of the Lamiaceae family against pathogenic and beneficial bacteria / Composición y actividad antibacteriana de aceites esenciales obtenidos de plantas de la familiaLamiaceae contra bacterias patógenas y benéficas / Composição e atividade antibacterina de azeites essenciais obtidos de plantas da família Lamiaceae contra bactérias patogênicas e benéficas

The qualitative composition and antibacterial activity of six essential oils obtained from plants cultivated in the Colombian Andes (Mentha spicata, Mentha piperita, Ocimum basilicum, Salvia officinalis, Rosmarinus officinalis and Thymus vulgaris) and a commercial essential oil of Origanum vulgare subsp. hirtum were investigated. The essential oil composition was determined by gas chromatography-mass spectrometry (GC-MS), while the antibacterial activity of the essential oils against Escherichia coli, Salmonella enteritidis, Salmonella typhimurium, Lactobacillus acidophilus and Bifidobacterium breve was measured as the minimum bacte icidal concentration (MBC) using the agar dilution method. The chemical analysis revealed the presence of 16-28 compounds in each oil, corresponding mainly to phenols, oxygenated and hydrocarbon monoterpenes. O. vulgare and T. vulgaris oils were active at low MBCs (MBC ≤ 5 mg/ml) against all bacteria evaluated, including beneficial microorganisms. In contrast, O. basilicum oil was more active against pathogenic bacteria (MBCs ≤ 10mg/ml) than beneficial bacteria (MBCs of 80 mg/ml). The present study shows that the antimicrobial potential of essential oils depends not only on the chemical composition of the oil but also on the targeted microorganism. This has important practical implications for essential oils intended to be used as feed additives with antibacterial properties for animal nutrition or pharmaceutical products with natural compounds.

Se investigó la composición cualitativa y la actividad antibacteriana de seis aceites esenciales obtenidos de plantas cultivadas en los Andes Colombianos (Mentha spicata, Mentha piperita, Ocimum basilicum, Salvia officinalis, Rosmarinus officinalis y Thymus vulgaris) y un aceite esencial comercial de Origanum vulgare subsp. hirtum. La composición de los aceites esenciales fue determinada por cromatografía de gasesespectrofotometría de masas (CG-EM), mientras que la actividad antibacteriana de los aceites esenciales contra Escherichia coli, Salmonella enteritidis, Salmonella typhimurim, Lactobacillus acidophilus y Bifidobacterium breve, fue medida como la concentración mínima bactericida (CMB) usando el método de dilución en agar. Los análisis químicos revelaron la presencia de16 - 28 compuestos en cada aceite, correspondiendo principalmente a monoterpenos fenolicos, oxigenados e hidrocarbonos. Los aceites de O. vulgare y T. vulgaris fueron activos contra todas las bacterias evaluadas, incluyendo microorganismos benéficos a CMBs bajas (CMB ≤ 5 mg/ml). En contraste, el aceite de O. basilicum fue más activo contra bacterias patógenas (CMBs ≤ 10 mg/ml) en comparación de bacterias benéficas (CMBs de 80 mg/ml). El presente estudio demostró que el potencial antimicrobiano de los aceites esenciales no depende solo de la composición química del aceite sino también del microorganismo por sí mismo. Estos resultados tienen implicaciones prácticas para los aceites esenciales usados como aditivos alimenticios con propiedades antibacterianas para la nutrición animal o productos farmacéuticos con compuestos naturales.

Pesquisou-se a composição qualitativo e a atividade antibacteriana de seis azeites essenciais obtidos de plantas cultivadas nos Andes Colombianos (Mentha spicata, Mentha piperita, Ocimum basilicum, Salvia officinalis, Rosmarinus officinalis e Thymus vulgaris) e um azeite essencial comercial de Origanum vulgare subsp. hirtum. A composição dos azeites essenciais foi determinada por cromatografía de gases -espectrofotometría de massas (CM-EM), enquanto a atividade antibacteriana dos azeites essenciais contra Escherichia coli, Salmonella enteritidis, Salmonella typhimurim, Lactobacillus acidophilus e Bifidobacterium breve foi medida como a concentração mínima bactericida (CMB) usando o método de diluição em ágar. As análises químicas revelaram a presença de16 - 28 compostos em cada azeite, correspondendo principalmente à monoterpenos fenólicos, hidrocarbonetos e oxigenados. Os azeites de O. vulgare e T. vulgaris foram ativos contra todas as bactérias testadas, incluindo microorganismos benéficos a CMBs baixas (CMB ≤ 5 mg/ml). Em contraste, o azeite de O. basilicum foi mais ativo contra bactérias patogénicas do que bactérias benéficas (CMBs de 80 mg/ml). Este estudo demonstrou o potencial antimicrobiano dos azeites essenciais depende da composição química do azeite e o microorganismo próprio. Estes resultados têm implicações práticas para os azeites essenciais usados como aditivos alimentícios com propriedades antibacterianas para a nutrição animal ou produtos farmacêuticos com produtos naturais.

Uptake and transformation of soil [14C]-trinitrotoluene by cool-season grasses.

This study investigated the fate and uptake of [(14)C]-TNT from soil into orchardgrass (Dactylis glomerata), perennial ryegrass (Lolium perenne), and tall fescue (Festuca arundinacea) over a one year period in a greenhouse-controlled environment. Pots (n = 4 for each grass, containing 10 mg cold TNT/kg soil + 1.2 mg [(14)C]-TNT/kg soil and controls with no TNT) were exposed to light and temperature conditions typical of June at 45 degrees N for 369 days. Three plant harvests were made (63, 181, and 369 days), and soil and plant materials were monitored for [(14)C]-TNT and metabolite concentrations. The 11.2 mg/kg TNT dose was not phytotoxic to the plant species tested. Continual uptake of TNT into grass blades was observed over the one-year period, with a total accumulation of 1.3%, 0.9%, and 0.8% of the initial soil [(14)C]-TNT dose for orchard grass, perennial ryegrass, and tall fescue, respectively. All [(14)C]-TNT residue in plant material was incorporated as bound residue. At final harvest, radioactivity was concentrated most highly in the root > crown > blade for all species. Soil TNT was gradually reduced to aminodinitro-toluenes and then further to an unidentified metabolite(s). Overall, orchardgrass appeared to be the most efficient species at taking up TNT.

A chemical marker proposal for the Lantana genus: composition of the essential oils from the leaves of Lantana radula and L. canescens.

Essential oil extracts from the leaves of two Lantana species (L. radula Sw. and L. canescens Kunth), for which no prior analysis has been reported, were analyzed by GC-MS. This information was utilized to propose chemical markers for Lantana species so that identification between physically similar plant species can be achieved through chemical analysis. Results showed 33 constituents for L. canescens, among which beta-caryophyllene (43.9%), beta-cubebene (10.1%), elixene (8.6%), beta-phellandrene (6.1%), alpha-caryophyllene (2.6%) and dehydro-aromadendrene (2.6%) were the principle components. L. radula revealed the presence of 21 compounds, the most abundant of which were beta-cubebene (31.0%), beta-caryophyllene (20.8%), elixene (10.0%), alpha-salinene (6.4%), beta-phellandrene (6.1%), copaene (4.9%) cadinene (1.4%) and psi-limonene (1.4%). The high concentration of beta-caryophyllene in the samples tested here and those in the literature make it a good candidate for a chemical marker for Lantana species, with beta-cubebene, elixene and beta-phellandrene following as minor compounds identified more sporadically in this genus. On the other hand, Lippia species, which are morphologically similar to those from the Lantana genus, would contain limonene, citral, carvacrol, beta-myrcene, camphor and thymol as the main chemical markers. These chemical markers would be a powerful tool for maintaining quality control in the extraction of essential oils for use in medicinal applications, as well as in identification of plant specimens to a taxonomist.

Acute aquatic toxicity of western juniper (Juniperus occidentalis) foliage and Port Orford cedar (Chamaecyparis lawsoniana) heartwood oils.

Recently, interest has developed for using essential oils from Western juniper (Juniperus occidentalis) foliage and Port Orford cedar (Chamaecyparis lawsoniana) heartwood in commercial products such as pest repellents and cosmetics. In order to gauge the relative toxicological risk that these oils pose to freshwater and marine organisms, the acute aquatic toxicity of these oils was evaluated using OPPTS guidelines to the cladoceran Daphnia magna, the rainbow trout Oncorhynchus mykiss and the green alga Selenastrum capricornutum. For western juniper foliage oil, no toxicity was exhibited toward D. magna or O. mykiss, even at 5.0 mg/L (the highest concentration tested and limit of solubility). For toxicity to S. capricornutum using algal cell density, the 72 and 96 h EC50 value was 1.7 mg/L and the no observable effect concentration (NOEC) was 0.63 mg/L. For Port Orford cedar heartwood oil, no toxicity was exhibited toward O. mykiss or S. capricornutum, even at 5.0 mg/L (the highest concentration tested and limit of solubility). The 48-h D. magna EC50 value was 1.9 mg/L; the NOEC values for algal cell density were 1.25 mg/L (72 h) and 0.63 mg/L (96 h). In summary, this study shows that western juniper foliage and Port Orford cedar heartwood oils demonstrate little to no risk to aquatic organisms.

Comparison of hepatic in vitro metabolism of the pyrrolizidine alkaloid senecionine in sheep and cattle.

OBJECTIVE: To compare hepatic metabolism of pyrrolizidine alkaloids (PAs) between sheep and cattle and elucidate the protective mechanism of sheep. SAMPLE POPULATION: Liver microsomes and cytosol from 8 sheep and 8 cattle. PROCEDURE: The PA senecionine, senecionine N-oxide (nontoxic metabolite) and 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP; toxic metabolite) were measured in microsomal incubations. The kcat (turnover number) was determined for DHP and N-oxide formation. Chemical and immunochemical inhibitors were used to assess the role of cytochrome P450s, flavin-containing monooxygenases (FMOs), and carboxylesterases in senecionine metabolism. The CYP3A, CYP2B, and FMO concentrations and activities were determined, in addition to the role of glutathione (GSH) in senecionine metabolism. RESULTS: DHP concentration did not differ between species. Sheep formed more N-oxide, had higher N-oxide kcat, and metabolized senecionine faster than cattle. The P450 concentrations and isoforms had a large influence on DHP formation, whereas FMOs had a large influence on N-oxide formation. In cattle, CYP3A played a larger role in DHP formation than in sheep. FMO activity was greater in sheep than in cattle. Addition of GSH to in vitro microsomal incubations decreased DHP formation; addition of cytosol decreased N-oxide formation. CONCLUSIONS AND CLINICAL RELEVANCE: Hepatic metabolism differences alone do not account for the variation in susceptibility seen between these species. Rather, increased ruminal metabolism in sheep appears to be an important protective mechanism, with hepatic enzymes providing a secondary means to degrade any PAs that are absorbed from the rumen.