Background: Ondansetron, a 5-hydroxytryptamine (5-HT) receptor antagonist, is generally regarded as a safe and well tolerated antiemetic. Meanwhile, some reports mentioned that it is a probable cause of single generalized seizures after intravenous administration. Our report may be the first to indicate repeated generalized seizures after intramuscular therapeutic dose of ondansetron. Methods and Results: We report a 24-year-old female with nausea and vomiting related to gastritis that experienced repeated ondansetron-induced seizures shortly after a single intramuscular therapeutic dose. Two minutes after intramuscular injection of 4 mg ondansetron, our patient developed the first generalized seizure. Within the following two hours, seizures occurred two more times. In the emergency department, the patient developed a fourth, but weaker and shorter, generalized seizure. The patient was not hypoglycemic, but her blood hemoglobin and serum electrolytes were below normal. A few hours later, the patient was discharged. The dramatic onset of the seizures, as well as the complete recovery and absence of any neurological sequel in our patient, indicated that it was probably related to ondansetron. Conclusion: Patients should be informed about the potential side effects of ondansetron especially the life-threatening repeated generalized seizures, and clinicians should restrict its use to hospitalized patients.
Our work has identified a group of oscillatory cell surface oxidases in the yeast Saccharomyces cerevisiae [1,2] classified as ECTO-NOX (ENOX) proteins with human homologs [
In this report, we identify an oscillatory, superoxidegenerating NADH oxidase activity with a period length of 26 min of S. cerevisiae. The ENOX3 in yeast was overexpressed by a selection process using a deletion library. One deletion strain had a deletion at the ENOX3 protein sequence and, thus, only showed yeast ENOX1 characteristics. The other deletion strain had a deletion at the yeast ENOX1 sequence and, thus only showed the ENOX3 characteristics. Using this approach, a candidate yeast gene potentially encoding a yeast ENOX3 protein with a 26 min period capable of generating superoxide was identified and characterized.
Candidates for yeast ENOX3 were selected from a deletion library of nonessential yeast genes which showed altered peak patterns from 7 - 8 to 5 peaks (
the formation of superoxide (Results).
Yeast were grown at room temperature with shaking in rich media (YEPD) for 1 - 2 days until saturation. The yeast strains were maintained on YEPD agar plates and stored at 4˚C. Yeast cells were inactivated by heating for 1 h at 70˚C prior to assay.
The yeast whole cell pellet with overexpression of Histagged YER113C, the putative yeast ENOX3, was resuspended in 20 mM Tris-HCL, pH 8.0 with 0.5 mM benzamidine, 0.5 mM PMSF and 1 mM 6-aminohexanoic acid. Cells were lysed by three passages through a French pressure cell at 20,000 psi. The resultant pellet was extracted sequentially. The supernatant following centrifugation at 10,000 rpm for 15 min at 40˚C was saved. The resultant pellet was then extracted with 20 mM Tris-HCL, pH 8.0, containing 1% Triton X-100, 20 mM Tris-HCL, pH 8.0, with 0.3% sarcosine, and in 20 mM Tris-HCL, pH 8.0 with 0.4% SDS. The combined supernatants were analyzed by SDS-PAGE and western blot and silver stain to determine the location of the yeast ENOX3. Additionally the supernatant was applied to a nickel nitrilotriacetic acid (Ni-NTA) column to bind the histidine tag and eluted with imidazole to further purify the protein. The identity of the YER113C protein was confirmed by sequencing.
The SDS-PAGE gels were sliced every 0.5 cm and the slices were eluted with 50 mM Tris-MES, pH 7.0 overnight in 4˚C. The gel slice eluates were assayed for ENOX activity based on NADH oxidation. Proteins were determined by the bicinchoninic acid (BCA) method with bovine serum albumin as a standard [
Yeast samples resolved on 10% SDS polyacrylamide gels were transferred to a nitrocellulose membrane at 90 V for 1 h. A 5% solution of non-fat milk powder was used for blocking and the probe used for the western blot was a 1:2500 anti-histidine antibody (Sigma A5588).
The oxidation of NADH was measured from the disappearance of NADH at a wavelength of 340 nm in a reaction mixture containing 50 mM Tris-MES pH 7.0, 2 mM KCN to inhibit mitochondrial oxidase activity, and 150 μM NADH, and YER113C sample at 37˚C with temperature control and stirring [
Measurements of superoxide production were based on a standard method where reduction of ferricytochrome c by superoxide was monitored from the increase in absorbance at 550 nm with reference at 540 nm [
The oxidation of reduced coenzyme Q10 (CoQ10H2) was determined using a Hitachi U-3110 spectrophotometer from the disappearance of reduced CoQ at both 290 nm and 410 nm [
YER113C samples were added to 2.5 ml of reaction buffer (50 mM Tris-Mes, pH 7.0). The reaction was preincubated with 0.5 µmol of 2.2’-dithiodipyridine (DTDP) in 5 µl DMSO. After 10 min of incubation, a further 3.5 µmol of DTDP were added in 35 µl of DMSO to start the reaction. The increase in absorbance due to the cleavage of DTDP was monitored at 340 nm. The specific activity of the cleavage reaction was calculated using a millimolar absorption coefficient of 6.21 [
As YEPD medium is fluorescent, heat-inactivated yeast cells were washed with PBS by centrifuging the cells in the benchtop mini centrifuge (VWR, Pennsylvaia) for 1 - 2 min, the supernatant was discarded and the pellet was resuspended in PBS. This suspension was used for measurements of NADH fluorescence.
The inactivated yeast suspension in PBS was added to wells of a black 96-well plate at a dilution of 1:10 in a volume of 20 µl. The PBS for the experiment was supplemented with 2% glucose to support enzyme activity. The plate was loaded into a Fluoroskan fluorescent plate reader and the samples were measured once every min for 2 - 8 h, with excitation at 355 nm and emission at 460 m. Data were analyzed by Fast Fourier Transform (Minitab 15) and decomposition analysis (Minitab 15) [
The induced yeast lysate when assayed for NADH oxidase activity (
peak was eliminated by the addition of superoxide dismutase to verify its being attributed to superoxide.
YER113C encodes a 706 aa protein of molecular weight 81,545 and isoelectric point pH 7.39 (
Functional motifs in common with mammalian arNOX proteins included a 683GLGALS (GXGXXS) adenine binding motif and 576YVY and 590YFY putative copper binding motifs as well as a potential disulfide
interchange site 557CGIYLC (CXXXXC). Also present was a conserved CQ/CE motif common to the mammalian ENOX3 family of proteins at 125CE.
Sliced and pulverized SDS-PAGE gels from which the YER113C proteins were eluted overnight in assay buffer evealed enzymatic activity in the region of the gel capable of superoxide generation based on reduction of ferricytochrome c. The active fraction of resolubilized pelleted material from the yeast lysate corresponded to a molecular weight of about 81.5 kDa (
The protein disulfide-thiol interchange activity of the gel slice elute containing 55 kDa truncation was determined from the cleavage of dithiodipyridine substrate (
The yeast activity was resistant to simalikalactone D, a
specific inhibitor of ENOX1 nor were the activity patterns phased by the addition of melatonin. Phasing by melatonin is an ENOX1 characteristic [2,4]. The period length, however, was increased to about 32 min by assay in D2O in place of water (
A specific ENOX3 inhibitor mixture of dormin + Schizandra + salicin [to 2.5 ml of assay volume were added 60 µl of an aqueous mixture of 4 mg/ml Schizandra (Schizandra chinensis extract, 9% schizandrins, Draco, San Jose, CA) plus 1 mg/ml salicin (Sigma, St. Louis, MO) and 20 µl of IBR Dormin (Israli Biotechnology Research, Ramat-Gan, Israel) = AgeLoc (NuSkin Enterprises, Provo, UT)] inhibited the activity by >90%. Also inhibitory were a herbal hot water infusion of dried savory at a concentration of 125 mg/ml (>90%), gallic acid (200 µM), and coenzyme Q10. The ENOX1-specific
NOX inhibitor simalikalactone D, the mammalian ENOX3 inhibitor tyrosol (
Based on DNA sequence, YER113C, the human TM9SF4 homolog was expressed in E. coli and shown to fulfill the requirements for an age-related ENOX (arNOX or ENOX3) protein. The pattern of activity oscillations consisted of 5 unequally spaced maxima with the requisite 26 min period length and the generation of superoxide. Superoxide generation was evidenced by the superoxide dismutase inhibited reduction of ferricytochrome c. Superoxide is not a reaction product of the two other ENOX proteins: ENOX1 with a 24 min period length [
The human ENOX3 cDNAs all encode polypeptides having a highly hydrophobic C-terminal portion were organized into 9 transmembrane regions [14,15]. The transmembrane domains all have a similar structure and sequence to form a novel family of multispanning domain proteins designated “TM9SF” (transmembrane protein 9 superfamily) by the Human Gene Nomenclature Committee. The leader member of the TM9SF family is the S. cerevisiae EMP70 gene product, a 70 kDa precursor that is processed into a 24 kDa protein (p24a) located in the endosomes [
There are 5 TM9 super family members known in the human genone (1 with two transcript variants, 2, 3 and 4).
The two transcript variants of human family member 1 are similar with the exception that number 1a transcript variant contains additional C-terminal residues absent from transcription variant 1b. The yeast homolog most closely resembles family member 4. Interestingly, the human TM9SF4 is highly expressed in human melanoma cells [
We thank Debby Parisi for assistance and Peggy Runck for manuscript preparation.
BCA: bicinchoninic acid;
ENOX3: age-related NADH oxidase;
CAPS: 3(cyclohexylamino)-1-propane sulfonic acid;
DTDP: dithiodipyridine;
ELISA: enzyme-linked immunosorbent assay;
ENOX: ECTO-NOX;
GSH: reduced glutathione;
Ni-NTA: nickel nitrilotriacetic acid;
IPTG: isopropyl-beta-D-thiogalactopyranoside;
PMSF: phenylmethylsulfonyl fluoride;
SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis;
SF: superfamily;
SOD: superoxide dismutase;
TFA: trifluoroacetic acid;
TM: transmembrane.