Worldwide dissemination of acquired carbapenem-hydrolysing class D β-lactamases in Acinetobacter spp. other than Acinetobacter baumannii

The aim of this study was to identify acquired OXA-type carbapenemases in Acinetobacter spp. other than Acinetobacter baumannii. From a total of 453 carbapenem-susceptible and -resistant Acinetobacter isolates collected worldwide, 23 were positive for bla_OXA genes by multiplex PCR. These isolates were identified as Acinetobacter pittii (n = 18), Acinetobacter nosocomialis (n = 2), Acinetobacter junii (n = 1) and Acinetobacter genomic species 14TU/13BJ (n = 2). The bla_OXA genes and associated insertion sequence (IS) elements were sequenced by primer walking. In 11 of these isolates, sequencing of the PCR products revealed that they were false-positive for bla_OXA. The remaining 12 isolates, originating from Europe, Asia, South America, North America and South Africa, harboured OXA-23 (n = 4), OXA-58 (n = 5), OXA-40-like (n = 1) and OXA-143-like (n = 1); one A. pittii isolate harboured both OXA-23 and OXA-58. IS elements were associated with bla_OXA in 10 isolates. OXA multiplex PCR showed a high degree of false-positive results (47.8%), indicating that detection of bla_OXA in non-baumannii Acinetobacter spp. should be confirmed using additional methods.     

Prevalence and characterisation of CTX-M β-lactamases amongst Escherichia coli isolates from healthy food animals in China

The impact of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae of food animal origins on human health has caught considerable attention worldwide. Intestinal Escherichia coli obtained from healthy food animals (pigs, cattle and poultry) in China were tested for the presence of ESBL genes. CTX-M-producing isolates were further characterised by pulsed-field gel electrophoresis (PFGE), phylogenetic grouping, genetic environment analysis, conjugation and plasmid replicon typing. A total of 127 of the 896 E. coli isolates showed reduced susceptibility to cefotaxime (minimal inhibitory concentration≥2 μg/mL). bla(CTX-M) genes were detected in 111 of the 127 isolates. The most common CTX-M types were CTX-M-14 (n=40), CTX-M-55 (n=29) and CTX-M-65 (n=22), followed by CTX-M-27, -15, -98, -24, -3, -102 and -104. CMY-2 was detected in two isolates. High clonal diversity was found amongst CTX-M-producing isolates. Insertion sequence ISEcp1 was observed 42 bp upstream of the start codon of all CTX-M-9 group genes, whereas the spacer region between the right inverted repeats and CTX-M-1 group genes varied from 45 bp to 127 bp. Most bla(CTX-M) genes were transferable by conjugation. IncFII, IncI1, IncFIB, IncN and IncA/C replicons were detected in 28, 21, 7, 5 and 1 of the 70 transconjugants carrying bla(CTX-M), respectively. This study demonstrates that commensal E. coli from healthy food animals can be important reservoirs of bla(CTX-M) genes and may contribute to the dissemination and transfer of these β-lactamase genes throughout China.     

Transferable plasmid-mediated quinolone resistance in association with extended-spectrum β-lactamases and fluoroquinolone-acetylating aminoglycoside-6'-N-acetyltransferase in clinical isolates of Vibrio fluvialis

Vibrio fluvialis, which causes cholera-like diarrhoea in humans, is one of the aetiological agents of acute diarrhoea in Kolkata, India, and is resistant to many antimicrobial agents. Two V. fluvialis isolates resistant to fluoroquinolones and β-lactam antimicrobials were found to have mutations in the quinolone resistance-determining regions (QRDRs) of GyrA at position 83 and of ParC at position 85 as well as carrying a 150 kb plasmid harbouring the quinolone resistance gene qnrA1, the ciprofloxacin-modifying enzyme-encoding gene aac(6′)-Ib-cr and genes encoding for extended-spectrum β-lactamases such as bla_SHV and bla_CTX-M-3. When this large plasmid was transferred to Escherichia coli by conjugation, the transconjugants showed a 10-75-fold increase in the minimum inhibitory concentrations of ciprofloxacin and norfloxacin. The qnrA1 gene was identified in a complex sul1-type integron in a plasmid of the transconjugants. Southern hybridisation and sequence analysis of qnrA1 and its flanking regions confirmed the presence of aac(6′)-Ib-cr and bla_CTX-M-3 but these were not associated with the sul1-type integron. Pulsed-field gel electrophoresis (PFGE) revealed that the two V. fluvialis isolates belonged to different clones. Although the presence of many qnr alleles has been reported amongst enteric bacteria in Asian countries, this is the first report on the emergence of qnrA1 in India. qnrA1 along with aac(6′)-Ib-cr and bla_CTX-M-3 genes on a mobile plasmid may spread to other bacterial species that are under the selective pressure of fluoroquinolones and β-lactam antimicrobials in this region.     

OprD mutations and inactivation, expression of efflux pumps and AmpC, and metallo-β-lactamases in carbapenem-resistant Pseudomonas aeruginosa isolates from South Korea

During a polymerase chain reaction (PCR)-based surveillance study of β-lactam resistance, 19 OXA-48-positive enterobacterial isolates were detected at nine Belgian hospitals from January 2010 to April 2011. Most cases were presumed to have been locally acquired and were detected in patients who had not travelled abroad. Clonally related outbreaks occurred in two different cities. The majority of isolates co-produced several β-lactamases as well as non-β-lactam resistance genes. This report highlights the rapid emergence and spread of OXA-48-producing Enterobacteriaceae in Belgium.     

PDE3, but not PDE4, reduces β1- and β2-adrenoceptor-mediated inotropic and lusitropic effects in failing ventricle from metoprolol-treated patients

Background and Purpose

PDE3 and/or PDE4 control ventricular effects of catecholamines in several species but their relative effects in failing human ventricle are unknown. We investigated whether the PDE3-selective inhibitor cilostamide (0.3–1 μM) or PDE4 inhibitor rolipram (1–10 μM) modified the positive inotropic and lusitropic effects of catecholamines in human failing myocardium.

Experimental Approach

Right and left ventricular trabeculae from freshly explanted hearts of 5 non-β-blocker-treated and 15 metoprolol-treated patients with terminal heart failure were paced to contract at 1 Hz. The effects of (-)-noradrenaline, mediated through β₁ adrenoceptors (β₂ adrenoceptors blocked with ICI118551), and (-)-adrenaline, mediated through β₂ adrenoceptors (β₁ adrenoceptors blocked with CGP20712A), were assessed in the absence and presence of PDE inhibitors. Catecholamine potencies were estimated from –logEC₅₀s.

Key Results

Cilostamide did not significantly potentiate the inotropic effects of the catecholamines in non-β-blocker-treated patients. Cilostamide caused greater potentiation (P = 0.037) of the positive inotropic effects of (-)-adrenaline (0.78 ± 0.12 log units) than (-)-noradrenaline (0.47 ± 0.12 log units) in metoprolol-treated patients. Lusitropic effects of the catecholamines were also potentiated by cilostamide. Rolipram did not affect the inotropic and lusitropic potencies of (-)-noradrenaline or (-)-adrenaline on right and left ventricular trabeculae from metoprolol-treated patients.

Conclusions and Implications

Metoprolol induces a control by PDE3 of ventricular effects mediated through both β₁ and β₂ adrenoceptors, thereby further reducing sympathetic cardiostimulation in patients with terminal heart failure. Concurrent therapy with a PDE3 blocker and metoprolol could conceivably facilitate cardiostimulation evoked by adrenaline through β₂ adrenoceptors. PDE4 does not appear to reduce inotropic and lusitropic effects of catecholamines in failing human ventricle.

Linked Article

This article is commented on by Eschenhagen, pp 524–527 of this issue. To view this commentary visit http://dx.doi.org/10.1111/bph.12168     

Phosphodiesterases do not limit β1-adrenoceptor-mediated sinoatrial tachycardia: evidence with PDE3 and PDE4 in rabbits and PDE1–5 in rats

The mammalian heart expresses at least five phosphodiesterases (PDE1–5). Catecholamines produce surges of inotropically relevant cAMP through β₁-adrenoceptor stimulation. cAMP is mainly hydrolysed by PDE3 and/or PDE4 thereby blunting contractility. Basal sinoatrial beating rate in mouse, rat, piglet and rabbit sinoatrial cells is reduced by PDE3 and/or PDE4 through hydrolysis of cAMP. However, in rodents, the tachycardia elicited by catecholamines through production of cAMP by β-adrenoceptor activation is not controlled by PDE3 and PDE4, despite a blunting effect of PDE3 or/and PDE4 on basal sinoatrial beating, but it is unknown whether PDE3 limits catecholamine-evoked tachycardia in the rabbit. Since rabbit sinoatrial cells are an important model for pacemaker research, we investigated whether the positive chronotropic effects of (?)-noradrenaline on spontaneously beating right atria of the rabbit are potentiated by inhibition of PDE3 with cilostamide (300 nM). We also studied the sinoatrial effects of the PDE4 inhibitor rolipram (10 μM) and its influence on the responses to (?)-noradrenaline. For comparison, we investigated the influence of cilostamide and rolipram on the positive inotropic responses to (?)-noradrenaline on rabbit left atria and right ventricular papillary muscles. Cilostamide and concurrent cilostamide?+?rolipram, but not rolipram alone, increased sinoatrial rate by 15% and 31% of the effect of (?)-isoprenaline (200?µM) but the PDE inhibitors did not significantly change the chronotropic potency of (?)-noradrenaline. In contrast in papillary muscle, the positive inotropic effects of (?)-noradrenaline were potentiated 2.4-, 2.6- and 44-fold by cilostamide, rolipram and concurrent cilostamide?+?rolipram, respectively. In left atrium, the positive inotropic effects of (?)-noradrenaline were marginally potentiated by cilostamide, as well as potentiated 2.7- and 32-fold by rolipram and by concurrent cilostamide and rolipram respectively. To compare the influence of PDE1–5 on basal sinoatrial rate and (?)-noradrenaline-evoked tachycardia, we investigated on rat right atria the effects of selective inhibitors. The PDE4 inhibitor rolipram and non-selective inhibitor isobutyl-methylxanthine caused tachycardia with –logEC₅₀s of 7.2 and 5.0 and E _max of 18% and 102% of (?)-isoprenaline, respectively. Rolipram did not change the chronotropic potency of (?)-noradrenaline. At high concentrations (10–30?µM), the PDE1, PDE3 and PDE5 inhibitors 8-methoxymethyl-3-isobutyl-1-methylxanthine, cilostamide and sildenafil, respectively, caused marginal tachycardia but did not significantly change the chronotropic potency of (?)-noradrenaline. The PDE2-selective inhibitor erythro-9-[2-hydroxy-3-nonyl]adenine caused marginal bradycardia at 30?µM and tended to reduce the chronotropic potency of (?)-noradrenaline. Rabbit PDE3 reduces basal sinoatrial rate. Although PDE4 only marginally reduces rate, under conditions of PDE3 inhibition, it further reduces sinoatrial rate. Both PDE3 and PDE4 control atrial and ventricular positive inotropic effects of (?)-noradrenaline. In contrast, neither PDE3 nor PDE4 limit the sinoatrial tachycardia induced by (?)-noradrenaline. In the rat, only PDE4, but not PDE1, PDE2, PDE3 and PDE5, reduces basal sinoatrial rate. None of the five rat PDEs limits the (?)-noradrenaline-evoked tachycardia. Taken together, these results confirm and expand evidence for our proposal that the cAMP-compartment modulating basal sinoatrial rate, controlled by PDE3 and/or PDE4, is different from the PDE-resistant cAMP compartment involved in β₁-adrenoceptor-mediated sinoatrial tachycardia.     

Differential outcomes from metabolic ratios in the identification of CYP2D6 phenotypes–focus on venlafaxine and O-desmethylvenlafaxine

Background  

Venlafaxine (VEN), a well accepted anti-depressant, is metabolized through the cytochrome P 450 (CYP) 2D6 isozyme to form O-desmethyvenlafaxine (ODV). Due to the involvement of CYP2D6, the formation of ODV is influenced by genetic polymorphism. We used standard tools of assessment to explore the phenotypic distribution in a retrospective manner using the pharmacokinetic (PK) data of VEN and ODV obtained from several bioavailability/bioequivalence (BA/BE) studies in healthy subjects using the reference formulation.     

Reversal of temperature-induced conformational changes in the amyloid-beta peptide,Aβ40, by the β-sheet breaker peptides 16–23 and 17–24

Background and purpose:

Aggregates of the protein amyloid-beta (Aβ) play a crucial role in the pathogenesis of Alzheimer''s disease (AD). Most therapeutic approaches to AD do not target Aβ, so determination of the factor(s) that facilitate aggregation and discovering agents that prevent aggregation have great potential therapeutic value.

Experimental approach:

We investigated ex vivo the temperature-sensitive regions of Aβ1–40 (Aβ40) and their interactions with octapeptides derived from sequences within Aβ40 –β-sheet breaker peptides (βSBP) – using enzyme-linked immunosorbent assay, and dot blot and far-UV circular dichroism (CD) spectroscopy. We measured changes within the physiological limits of temperature, using antibodies targeting epitopes 1–7, 5–10, 9–14 and 17–21 within Aβ40.

Key results:

Temperature-dependent conformational changes were observed in Aβ40 at epitopes 9–14 and 17–21 at 36–38 and 36–40°C respectively. The βSBPs 16–23 and 17–24, but not 15–22 and 18–25, could inhibit the changes. Moreover, βSBPs 16–23 and 17–24 increased digestion of Aβ40 by protease K, indicating a decreased aggregation of Aβ40, whereas βSBPs 15–22 and 18–25 did not increase this digestion. CD spectra revealed that β-sheet formation in Aβ40 at 38°C was reduced with βSBPs 16–23 and 17–24.

Conclusions and implications:

The epitopes 9–14 and 17–21 are the temperature-sensitive regions within Aβ40. The βSBPs, Aβ16–23 and 17–24 reversed temperature-induced β-sheet formation, and decreased Aβ40 aggregation. The results suggest that the 17–23 epitope of Aβ40 is crucially involved in preventing Aβ40 aggregation and consequent deposition of Aβ40 in AD brain.     

1-Deoxynojirimycin isolated from Bacillus subtilis improves hepatic lipid metabolism and mitochondrial function in high-fat–fed mice

The aim of this study was to determine whether 1-deoxynojirimycin (DNJ) isolated from Bacillus subtilis MORI beneficially influences lipid metabolism and mitochondrial function in the liver of mice fed a high-fat diet in addition to the anti-obesity properties of DNJ. Male C57BL/6 mice (n = 29; 5 weeks old) were randomly assigned to three groups: normal control diet (CTL, n = 10), high-fat diet (HF, n = 10), and high-fat diet supplemented with DNJ (DNJ, n = 9). After 12 weeks, the HF group exhibited higher overall weight gain, of the liver, and of various fat pads than the CTL and DNJ groups did. The HF group also showed greater expression of C/EBPα and CD36 mRNA in the liver than that in the CTL and/or DNJ groups. In addition, mRNA expressions of AAC and FAS were lower, while mRNA expression of PGC-1β was higher in the liver of the DNJ group than that of the HF group. The hepatic expression of p-AMPK/AMPK was higher in the DNJ group than in the HF group. This study provides novel insight into the protective effect of DNJ supplementation against obesity-induced hepatic lipid abnormalities and mitochondrial dysfunction.     

New dihydrochalcone glycosides from Lithocarpus litseifolius and the phenomenon of C–H→C–D exchange observed in NMR spectra of phenolic components

Two new dihydrochalcone glycosides named 6″-O-acetyltrilobatin (1) and 3″-O-acetylphloridzin (2) as well as four known compounds were isolated from the leaves of Lithocarpus litseifolius (Hance) Chun (family Fagaceae). Their structures were elucidated on the basis of spectroscopic analyses. The phenomena of C–H → C–D exchange were observed in NMR spectra of the isolated phenolic components when measured in deuterated methanol.     

Involvement of CDK4, pRB, and E2F1 in ginsenoside Rg_1 protecting rat cortical neurons from β-amyloid-induced apoptosis

AIM: To explore the possible mechanism of β-amyloid (Aβ)-induced apoptosis in rat cortical neurons and the protective effect of ginsenoside Rg_1. METHODS: AO-EB staining was used to quantify the apoptotic cells. DNA fragmentation was observed by gel electrophoresis. The levels of cyclin-dependent kinases-4 (CDK4) and phosphorylated pRB were detected by Western blot. RT-PCR was used to examine the expression of E2FI mRNA. RESULTS: Treatment with Aβ_(1-40) at the concentration of 20, 40, 80 mg/L for 48 h induced rat cortical neuron poptosis from 12.5％±1.5％(control) to 22.3％±1.4％, 38.8％±1.3％, 36.7％±1.4％, respectively. Pretreatment with Rg_1 at the dose of 0.5, 1, 2, 4, 8, 16 μmol/L for 24 h, then treatment with Aβ_(1-40)40 mg/L for 24 h, the percentage of apoptotic neurons decreased from 38.8％±1.3％ to 14.5％±1.3％, 13.3％±1.0％, 11.6％±0.29％, 11.8％±1.0％, 6.2％±0.8％, 5.8％±0.8％, respectively. After treatment with Aβ_(1-40)40 mg/L for 24 h, there were transient increases in CD     

Cyclin D1/cdk4, estrogen receptors α and β, in N-methyl-N'-nitro-N-nitrosoguanidine-induced rat gastric carcinogenesis: immunohistochemical study

Hyperproliferative cell growth due to cyclin D1/cdk4, marker of cellular proliferation, is considered to be regulated by the expression of estrogen receptors (ERs). We investigated the immunohistochemical expression of cyclin D1/cdk4 and ERs in N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced rat gastric carcinogenesis. The gastric cancer incidence and expression of cyclin D1/ckd4 in gastric carcinogenesis were significantly higher in males than females. Although the ERα expression index was similar in both sexes, the ERβ expression in preneoplastic hyperplastic lesions as well as gastric cancers was significantly higher in females than in males. The present study revealed a gender difference in MNNG-induced rat gastric carcinogenesis that seemed to involve the sex difference in cyclin D1/cdk4 expression, and ERβ expression became evident at the preneoplastic promotion stage in gastric carcinogenesis.     

Friendly, and not so friendly, roles of Rac1 in islet β-cell function: lessons learnt from pharmacological and molecular biological approaches

Glucose-stimulated insulin secretion [GSIS] involves a sequence of metabolic events leading to small G-protein [e.g., Rac1]-mediated cytoskeletal remodeling to promote granule mobilization toward the plasma membrane for fusion and release of insulin. Existing evidence supports a positive modulatory role for Rac1 in GSIS. Specific regulatory factors of Rac1 function, including the guanine nucleotide exchange factors [e.g., Tiam1] have also been identified and studied in the islet. Inhibition of Tiam1/Rac1 signaling axis attenuates GSIS suggesting its pivotal role in insulin secretion. In addition to its positive [i.e., friendly] roles in GSIS, Rac1 also plays “non-friendly” role[s] in the islet function. For example, it up-regulates the intracellular reactive oxygen species [ROS] levels via activation of phagocyte-like NADPH oxidase [Nox]. Despite the emerging evidence that a tonic increase in intracellular ROS is necessary for GSIS, experimental evidence also suggests that chronic exposure of β-cells to high glucose, palmitate or cytokines results in the onset of oxidative stress leading to reduction in mitochondrial membrane potential, cytosolic accumulation of cytochrome C and activation of caspase-3 leading to β-cell apoptosis. Pharmacological and molecular biological inhibition of Rac1 activation affords partial protection against Nox-induced oxidative stress and mitochondrial dysfunction induced by elevated glucose, lipids or cytokines. Herein, we overview the existing evidence to suggest positive as well as negative modulatory roles of Rac1 in islet function. Potential avenues for future research including development of inhibitors to halt the Rac1–Nox activation and generation of oxidative stress leading to the metabolic dysfunction of the β-cell are discussed.     

Dopamine D1 Receptor Activation Rescues Extinction Impairments in Low-Estrogen Female Rats and Induces Cortical Layer-Specific Activation Changes in Prefrontal–Amygdala Circuits

Post-traumatic stress disorder (PTSD) is twice as common in women as in men; it is a major public health problem whose neurobiological basis is unknown. In preclinical studies using fear conditioning and extinction paradigms, women and female animals with low estrogen levels exhibit impaired extinction retrieval, but the mechanisms that underlie these hormone-based discrepancies have not been identified. There is much evidence that estrogen can modulate dopaminergic transmission, and here we tested the hypothesis that dopamine–estrogen interactions drive extinction processes in females. Intact male and female rats were trained on cued fear conditioning, and received an intraperitoneal injection of a D1 agonist or vehicle before extinction learning. As reported previously, females that underwent extinction during low estrogen estrous phases (estrus/metaestrus/diestrus (EMD)) froze more during extinction retrieval than those that had been in the high-estrogen phase (proestrus; PRO). However, D1 stimulation reversed this relationship, impairing extinction retrieval in PRO and enhancing it in EMD. We also combined retrograde tracing and fluorescent immunohistochemistry to measure c-fos expression in infralimbic (IL) projections to the basolateral area of the amygdala (BLA), a neural pathway known to be critical to extinction retrieval. Again we observed diverging, estrous-dependent effects; SKF treatment induced a positive correlation between freezing and IL-BLA circuit activation in EMD animals, and a negative correlation in PRO animals. These results show for the first time that hormone-dependent extinction deficits can be overcome with non-hormone-based interventions, and suggest a circuit-specific mechanism by which these behavioral effects occur.     

Synthesis and preliminary in vitro activity of mono- and bis-1H-1,2,3-triazole-tethered β-lactam–isatin conjugates against the human protozoal pathogen Trichomonas vaginalis

In this study, we describe the synthesis of mono- and bis-1H-1,2,3-triazole-tethered β-lactam–isatin conjugates using copper-catalysed azide-alkyne cycloaddition reaction between mono- and di-propargylated azetidin-2-ones and N-alkylazido isatins. The synthesized conjugates were evaluated for their preliminary in vitro analysis against Trichomonas vaginalis at 50 μM. The efficacy of synthesized hybrids was observed to depend on the substituent at N-1 position of β-lactam ring, as well as the presence of single/double 1H-1,2,3-triazole linker. Among the synthesized conjugates, the presence of a p-tolyl substituent at N-1 of β-lactam ring was preferred for good activity profiles while the increase in spacer length did not influence the efficacy of the compounds. Compounds with high levels of potency were further analysed to determine their IC₅₀ values, as well as cytotoxicity profiles against mammalian cells. The most active compound in the synthesized conjugates displayed an IC₅₀ value of 10.49 μM against cultured G3 strain of T. vaginalis and was non-toxic to cultured mammalian HeLa cells at the same concentration.     

Involvement of the dopamine D1 receptor system in the anxiolytic effect of cedrol in the elevated plus maze and light–dark box tests

Cedrol, mainly derived from Juniperus virginiana L. essential oil, has been demonstrated the anxiolytic effect, although its mechanism of action is still not fully established. In the present study, male ICR mice were submitted to the elevated plus maze (EPM) and light–dark box (LDB) tests to investigate the putative mechanism of anxiolytic effect. WAY100635 (5-HT_1A receptor antagonist), flumazenil (benzodiazepine receptor antagonist), SCH23390 (dopamine D₁ receptor antagonist) or sulpiride (dopamine D₂/D₃ receptor antagonist) were used in the behavioral experiment to determine the mechanism of action of cedrol. Subsequently, the monoamine neurotransmitter levels were evaluated after behavioral tests. The data suggest that no significant effect in behavioral parameters were observed after sole intraperitoneal (i.p.) injection of antagonists compared to saline group. The anxiolytic effect of cedrol in behavioral procedures was blocked by either WAY100635 or flumazenil. The anxiolytic effect of cedrol (1200 mg/kg) was effectively antagonized by SCH23390 (0.125 mg/kg). Furthermore, cedrol decreased the DA and NE levels in hippocampus, striatum and hypothalamus. The present findings suggest that the dopaminergic system (D₁ receptor) rather than serotoninergic or GABAergic system may potentially be involved in the modulation of cedrol-induced anxiolytic-like behaviors in mice.     

Metabolism of 26,27-hexafluoro-1α,25-dihydroxyvitamin D3 and 26,27-hexafluoro1α,23(S)25-trihydroxyvitamin D3 in ROS17/2.8 cells transfected with a plasmid expressing CYP24

1. To clarify the possibility that the metabolism of 26,27-hexafl uoro-1α, 25-dihydroxyvitamin D₃ [F₆-1,25(OH)₂D₃] to 26,27-hexafluoro-1α,23(S),25-trihydroxyvitamin D₃ [F₆-1,23,25(OH)₃D₃ and that of F₆-1,23,25(OH)₃D₃ to 26,27-hexafluoro-23-oxo-1α,25-dihydroxyvitamin D₃ [F₆-23-oxo-1,25(OH)₂D₃] are catalysed by 25-hydroxyvitamin D 24-hydroxylase (CYP24), ROS17}2.8 cells transfected with a plasmid expressing CYP24 [pSVL-CYP24–] and a corresponding blank plasmid [pSLVCYP24R(®)] were used. 2. Incubation of [1β-³H]-F-1,25(OH)₂D₃ for 2 and 5 days with ROS17}2.8 cells transfected with pSVL-CYP24– generated a metabolite that co-migrated with authentic F₆ -1,23,25(OH)₃D₃ in both normal phase and reversed-phase HPLC systems. 3. Incubation of [1β-³H]-F₆ -1,23,25(OH)₃D₃ for 5 days with pSVL-CYP24– - transfected ROS 17}2.8 cells generated a metabolite that co-migrated with authentic F₆ -23-oxo-1,25(OH)₂D₃. In contrast, the metabolites F₆ -1,23,25(OH)₃D₃ or F₆ -23-oxo- 1,25(OH)₂D₃ were not generated in the cells transfected with pSVL-CYP24R(-). 4. The results indicate that CYP24 catalyses the conversion of F₆ -1,25(OH)₂D₃ to F₆ -1,23,25(OH)₃D₃ and that of F₆ -1,23,25(OH)₃D₃ to F₆ -23-oxo-1,25(OH)₂D₃.