Nutrient digestibility, rumen parameters, and (cannabinoid) residues in sheep fed a pelleted diet containing green hemp (Cannabis sativa L.) biomass.

The feeding value for ruminants of green hemp biomass, from the low Δ9-tetrahydrocannabinol (Δ9-THC) variety of Cannabis sativa L., is unknown. Twelve Merino ewes were individually penned and randomly allocated on a stratified liveweight basis to one of two pelleted dietary treatments, control (0% hemp, n = 6) or hemp (42% green hemp biomass, n = 6) that delivered a diet meeting the nutrient requirements of the animals. The experimental period consisted of 17 d dietary and housing adaptation, followed by 7 d total urine and feces collection for determination of apparent nutrient digestibility. A ruminal fluid sample was collected on day 27 and assessed for pH, ammonia, volatile fatty acid (VFA), and cannabinoid concentrations. A blood sample from the jugular vein and incisional subcutaneous fat biopsy from an area around the base of the tail were collected on day 28 with additional fat biopsies taken 35 d and 140 d post-feeding to measure cannabinoids. The dry matter (DM), organic matter (OM), and crude protein (CP) digestibilities, along with total VFA concentration did not differ (P = 0.713) between the two diets; however, acid detergent fiber (ADF) and neutral detergent fiber (NDF) digestibilities (P < 0.001), water intake (P = 0.023), and fecal water output (P < 0.001) were significantly lower for the sheep-fed Hemp. Rumen pH did not vary (P = 0.256) between diets, but ruminal ammonia concentration was significantly lower (P = 0.024) for sheep consuming Hemp. Sheep-fed Hemp had significantly greater molar proportions of butyric (P = 0.039) and hexanoic (P = 0.012) acids and lower molar proportions of propionic acid (P = 0.003). There were no differences between diets for N intake (P = 0.175), fecal N output (P = 0.253), and N balance (P = 0.695), with all sheep in positive N balance; however, there was significantly lower (P = 0.001) urinary N output for sheep-fed Hemp. Cannabidiolic acid (CBDA) and tetrahydrocannabinolic acid (THCA) were detected in plasma of all sheep-fed Hemp. ∆9-tetrahydrocannabinol was present in the subcutaneous fat of four of the six sheep on the final day of being fed Hemp, and in all (six) sheep 35 d post-feeding and one sheep 140 d post-feeding Hemp. No cannabinoids were detected in the corresponding samples taken from the sheep-fed Control. Thus, despite green hemp biomass being nutritionally a suitable feed for ruminants, under current Food Standards in Australia, the presence of these cannabinoid residues restricts its use in ruminant diets.

The influence of epileptic neuropathology and prior peripheral immunity on CNS transduction by rAAV2 and rAAV5.

Adeno-associated virus (AAV) provides a promising platform for clinical treatment of neurological disorders owing to its established efficacy and lack of apparent pathogenicity. To use viral vectors in treating neurological disease, however, transduction must occur under neuropathological conditions. Previous studies in rodents have shown that AAV5 more efficiently transduces cells in the hippocampus and piriform cortex than AAV2. Using the kainic acid (KA) model of temporal lobe epilepsy and AAV2 and 5 carrying a hybrid chicken ß-actin promoter driving green fluorescent protein (GFP), we found that limbic seizure activity caused substantial neuropathology and resulted in a significant reduction in subsequent AAV5 transduction. Nonetheless, this reduced transduction still was greater than AAV2 transduction in control rats. Although KA seizures compromise blood-brain barrier function, potentially increasing exposure of target tissue to circulating neutralizing antibodies, we observed no interaction between KA seizure-induced damage and immunization status on AAV transduction. Finally, while we confirmed the near total neuronal-specific transgene expression for both serotypes in control rats, AAV5-GFP expression was increasingly localized to astrocytes in seizure-damaged areas. Thus, the pathological milieu of the injured brain can reduce transduction efficacy and alter viral tropism- both relevant concerns when considering viral vector gene therapy for neurological disorders.

False-positive results on colorimetric carbon dioxide analysis in neonatal resuscitation: potential for serious patient harm.

A term infant requiring resuscitation was found to have a false-positive color change on a colorimetric carbon dioxide device as a result of administration of epinephrine via an endotracheal tube. Using models of direct application and vapor exposure with a test lung, we discovered that epinephrine, atropine, infasurf and naloxone may result in false-positive color change. This false-positive response may lead to delayed recognition of esophageal intubation.

G beta association and effector interaction selectivities of the divergent G gamma subunit G gamma(13).

G gamma(13) is a divergent member of the G gamma subunit family considered to be a component of the gustducin G-protein heterotrimer involved in bitter and sweet taste reception in taste bud cells. G gamma(13) contains a C-terminal asparagine-proline-tryptophan (NPW) tripeptide, a hallmark of RGS protein G gamma-like (GGL) domains which dimerize exclusively with G beta(5) subunits. In this study, we investigated the functional range of G gamma(13) assembly with G beta subunits using multiple assays of G beta association and G beta gamma effector modulation. G gamma(13) was observed to associate with all five G beta subunits (G beta(1-5)) upon co-translation in vitro, as well as function with all five G beta subunits in the modulation of Kir3.1/3.4 (GIRK1/4) potassium and N-type (alpha(1B)) calcium channels. Multiple G beta/G gamma(13) pairings were also functional in cellular assays of phospholipase C (PLC) beta 2 activation and inhibition of G alpha(q)-stimulated PLC beta 1 activity. However, upon cellular co-expression of G gamma(13) with different G beta subunits, only G beta(1)/G gamma(13), G beta(3)/G gamma(13), and G beta(4)/G gamma(13) pairings were found to form stable dimers detectable by co-immunoprecipitation under high-detergent cell lysis conditions. Collectively, these data indicate that G gamma(13) forms functional G beta gamma dimers with a range of G beta subunits. Coupled with our detection of G gamma(13) mRNA in mouse and human brain and retina, these results imply that this divergent G gamma subunit can act in signal transduction pathways other than that dedicated to taste reception in sensory lingual tissue.

Xenobiotic biotransforming enzymes in the central nervous system: an isoform of flavin-containing monooxygenase (FMO4) is expressed in rabbit brain.

The flavin-containing monooxygenase (FMO, EC 1.14.13.8) is involved in the metabolism of a number of important xenobiotics including many which affect the central nervous system (CNS). Recently, reports in the literature concerning the amount, activity, location, and isozyme characteristics of this enzyme in the brain have presented conflicting evidence. In order to resolve some of the controversy surrounding FMO in the brain, a highly sensitive method for the detection of flavin-containing monooxygenase (FMO) mRNA in whole brain was employed. A poorly conserved region of FMO transcripts was used to design five sets of oligonucleotide primers. Each primer set was specific for one of the five currently known isoforms of FMO. Four and five isoforms, respectively, are expressed in rabbit liver and kidney, as determined by reverse transcription-polymerase chain reaction. However, only one set of primers amplified a specific rabbit brain cDNA fragment. The sequence of the amplification produced affirmed its identity as a segment of FMO4 cDNA. Thus, the FMO of rabbit brain may consist of a single, as yet uncharacterized isozyme and, contrary to several recent reports, is likely to be expressed at low levels.

Pesticide-metabolizing enzymes.

Pesticides are known to function as substrates, inhibitors and inducers of drug-metabolizing enzymes, with the same compound frequently acting in more than one of these roles. Current studies of phase I metabolism of pesticides include cytochrome P450 (P450) and the flavin-containing monooxygenase (FMO), with particular reference to individual isozymes. In mouse liver, the level of FMO1 is gender dependent, FMO3 is gender specific, while FMO5 appears to be gender independent. The isozyme specificity of methylenedioxyphenyl synergists for induction of P450 in mouse liver involves P450s 1A1, 1A2 and 2B10, including a non-Ah receptor-dependent mechanism for 1A2 induction. The substrate specificity of mouse and human P450 and FMO isozymes is discussed.

Olfactory toxicity of methimazole: dose-response and structure-activity studies and characterization of flavin-containing monooxygenase activity in the Long-Evans rat olfactory mucosa.

Methimazole is a compound administered to humans for the treatment of hyperthyroidism and is used experimentally as a model substrate for the flavin-containing monooxygenase (FMO) system. Previous results from this laboratory demonstrated that methimazole is an olfactory system toxicant, causing nearly complete destruction of the olfactory epithelium in the male Long-Evans rat following a single ip dose of 300 mg/kg. The present studies were undertaken to determine the dose-response relationship for methimazole-induced olfactory mucosal damage and to determine whether or not similar damage occurs as a result of oral administration, mimicking the relevant route of human exposure. We also investigated the mechanism of olfactory toxicity of methimazole by means of a structure-activity study and began the characterization of the form(s) of FMO present in the olfactory mucosa of the male Long-Evans rat. Dose-response analysis demonstrated that methimazole causes olfactory mucosal damage at doses of 25 mg/kg ip and greater. The results of gavage studies showed that a single oral dose of 50 mg/kg also caused olfactory mucosal damage. Two structurally related compounds, methylimidazole and methylpyrrole, were not olfactory toxicants, suggesting that a reactive intermediate generated in the course of metabolizing methimazole to an S-oxide is the olfactory toxic species. Microsomal incubation studies revealed the presence of methimazole S-oxidation activity in olfactory mucosal microsomes at levels comparable to those in liver. An anti-mouse liver FMO antibody reacted on Western blots with olfactory mucosal microsomes. These findings demonstrate a dose-response for the olfactory toxicity of methimazole and suggest that characterization of human olfactory mucosal FMO activity may be necessary to assess the potential for human risk associated with therapeutic exposure to methimazole.

Gender differences in hepatic expression of flavin-containing monooxygenase isoforms (FMO1, FMO3, and FMO5) in mice.

Hepatic flavin-containing monooxygenase (FMO) activity of microsomes from adult CD-1, Swiss-Webster, C57BL/6, and DBA/2 mice was found to be significantly higher in females than in males. Based on protein and mRNA levels in CD-1 mice, FMO forms responsible for the gender difference in FMO activity were FMO1 and FMO3. FMO1 expression was two to three times higher in female mice compared with males; FMO3, however, which was expressed at levels equivalent to FMO1 in female mice, was not detected in males. The expression of FMO5 was approximately equal in both sexes. FMO2 and FMO4 transcripts were not evident in hepatic mRNA from mice. Protein and mRNA levels appear to be coregulated with regard to gender-selective or gender-specific expression of FMO1 or FMO3, respectively. FMO5, which demonstrates no gender-selective expression in mice, may be regulated by different mechanisms. Examination of protein levels among Swiss-Webster, C57BL/6, and DBA/2 strains revealed a gender-dependent expression of FMO isozymes identical to the CD-1 strain.

Metabolism of thioridazine by microsomal monooxygenases: relative roles of P450 and flavin-containing monooxygenase.

1. The metabolism of thioridazine by the flavin-containing monooxygenase (FMO) of mouse liver and several P450 isozymes was examined using microsomes, purified FMO, and expressed P450 isozymes. Metabolites were identified by hplc. 2. Thermal inactivation and antibodies to NADPH P450 reductase were used to selectively inactivate FMO and P450 respectively. Inactivation of FMO by heat-treatment reduced the formation of thioridazine-N-oxide and northioridazine, whereas inactivation of P450 resulted in decreased amounts of thioridazine-2-sulphoxide, northioridazine, and thioridazine-5-sulphoxide. 3. Liver microsomes from mouse induced with phenobarbital, 3-methylcholanthrene, or acetone were compared with control microsomes. Phenobarbital induction resulted in increased formation of all metabolites except thioridazine-N-oxide, while retaining a general metabolic profile similar to that achieved with control microsomes. Neither 3-methylcholanthrene nor acetone induction had any effect on the in vitro metabolism of thioridazine. 4. FMO purified from mouse liver produced thioridazine-N-oxide as the major metabolite. 5. Preliminary experiments with commercially prepared microsomes made from cells expressing recombinant human liver P450 2D6 and 3A4 suggested that thioridazine is metabolized by 2D6 but not 3A4.