Emetic responses to T-2 toxin, HT-2 toxin and emetine correspond to plasma elevations of peptide YY3-36 and 5-hydroxytryptamine.

Trichothecene mycotoxins are a family of potent translational inhibitors that are associated with foodborne outbreaks of human and animal gastroenteritis in which vomiting is a clinical hallmark. Deoxynivalenol (DON, vomitoxin) and other Type B trichothecenes have been previously demonstrated to cause emesis in the mink (Neovison vison), and this response has been directly linked to secretion of both the satiety hormone peptide YY3-36 (PYY3-36) and neurotransmitter 5-hydroxytryptamine (5-HT). Here, we characterized the emetic responses in the mink to T-2 toxin (T-2) and HT-2 toxin (HT-2), two highly toxic Type A trichothecenes that contaminate cereals, and further compared these effects to those of emetine, a natural alkaloid that is used medicinally and also well known to block translation and cause vomiting. Following intraperitoneal (IP) and oral exposure, all three agents caused vomiting with evident dose-dependent increases in both duration and number of emetic events as well as decreases in latency to emesis. T-2 and HT-2 doses causing emesis in 50 % of treated animals (ED50s) were 0.05 and 0.02 mg/kg BW following IP and oral administration, respectively, whereas the ED50s for emetine were 2.0 and 1.0 mg/kg BW for IP and oral exposure, respectively. Importantly, oral administration of all three toxins elicited marked elevations in plasma concentrations of PYY3-36 and 5-HT that corresponded to emesis. Taken together, the results suggest that T-2 and HT-2 were much more potent than emetine and that emesis induction by all three translational inhibitors co-occurred with increases in circulating levels of PYY3-36 and 5-HT.

Emetine and/or its metabolites are genotoxic in somatic cells of Drosophila melanogaster.

Emetine is one of the two active ingredients of syrup of ipecac which is used medicinally as antiparasitic and emetic, however little is known about its genotoxic activity. The goal of this study was to determine whether and how emetine and/or its metabolites might produce mitotic recombination using the in vivo Drosophila w/w+ eye somatic assay. A standard strain (which expresses basal levels of cytochrome P450 enzymes) and an insecticide-resistant strain (which constitutively over-expresses P450 genes) were employed. The results showed that emetine and/or its metabolites are active in the assay and that the genotoxic potential is significantly influenced in the presence of higher than normal concentrations of P450.

Apoptosis in early development of the sea urchin, Strongylocentrotus purpuratus.

Apoptosis provides metazoans remarkable developmental flexibility by (1) eliminating damaged undifferentiated cells early in development and then (2) sculpting, patterning, and restructuring tissues during successive stages thereafter. We show here that apoptotic programmed cell death is infrequent and not obligatory during early embryogenesis of the purple sea urchin, Strongylocentrotus purpuratus. During the first 30 h of urchin development, fewer than 20% of embryos exhibit any cell death. Cell death during the cleavage stages consists of necrotic or pathological cell death, while cell death during the blastula and gastrula stages is random and predominantly caspase-mediated apoptosis. Apoptosis remains infrequent during the late blastula stage followed by a gradual increase in frequency during gastrulation. Even after prolonged exposure during the cleavage period to chemical stress, apoptosis occurs in less than 50% of embryos and always around the pre-hatching stage. Embryonic suppression of apoptosis through caspase inhibition leads to functionally normal larvae that can survive to metamorphosis, but in the presence of inducers of apoptosis, caspase inhibition leads to deformed larvae and reduced survival. Remarkably, however, pharmacological induction of apoptosis, while reducing overall survival, also significantly accelerates development of the survivors such that metamorphosis occurs up to a week before controls.

Emetine inhibits glycolysis in isolated, perfused rat hearts.

This work was designed to test whether phosphofructokinase is a target for emetine action on the heart. The effects of 37 microM emetine on the activities of phosphofructokinase and hexokinase were measured in homogenates from perfused hearts. The action of increasing concentrations of emetine was determined in nonperfused heart homogenates. The effect of 37 microM emetine or control solutions on the concentration of fructose-6-phosphate and fructose- 1,6-phosphate was measured. The effect of 37 microM emetine or control perfusion on the utilization of fructose-6-phosphate by phosphofructokinase in centrifugation supernatants of homogenates and in reconstituted 27,000g pellets was measured. Double-reciprocal plots of fructose-6-phosphate concentrations vs phosphofructokinase activities were plotted. Emetine decreased phosphofructokinase activity in homogenates from both perfused and nonperfused hearts. Emetine did not inhibit cardiac hexokinase activity. In homogenates from nonperfused hearts, the maximal inhibition with high concentrations of emetine was approx 50%. Emetine perfusion caused a simultaneous increase in the phosphofructokinase substrate fructose-6-phosphate and a decrease in the phosphofructokinase product fructose-1,6-bisphosphate. Phosphofructokinase and, consequently, glycolytic flux appear to be subcellular targets for emetine in the heart. Homogenate centrifugation studies indicate that emetine acts on bound rather than unbound phosphofructokinase. The inhibition may be uncompetitive in nature.

Molecular determinants of apoptosis induced by the cytotoxic ribonuclease onconase: evidence for cytotoxic mechanisms different from inhibition of protein synthesis.

Cytotoxic endoribonucleases (RNases) possess a potential for use in cancer therapy. However, the molecular determinants of RNase-induced cell death are not well understood. In this work, we identify such determinants of the cytotoxicity induced by onconase, an amphibian cytotoxic RNase. Onconase displayed a remarkable specificity for tRNA in vivo, leaving rRNA and mRNA apparently undamaged. Onconase-treated cells displayed apoptosis-associated cell blebbing, nuclear pyknosis and fragmentation (karyorrhexis), DNA fragmentation, and activation of caspase-3-like activity. The cytotoxic action of onconase correlated with inhibition of protein synthesis; however, we present evidence for the existence of a mechanism of onconase-induced apoptosis that is independent of inhibition of protein synthesis. The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe) fluoromethyl ketone (zVADfmk), at concentrations that completely prevent apoptosis and caspase activation induced by ligation of the death receptor Fas, had only a partial protective effect on onconase-induced cell death. The proapoptotic activity of the p53 tumor suppressor protein and the Fas ligand/Fas/Fas-associating protein with death domain (FADD)/caspase-8 proapoptotic cascade were not required for onconase-induced apoptosis. Procaspases-9, -3, and -7 were processed in onconase-treated cells, suggesting the involvement of the mitochondrial apoptotic machinery in onconase-induced apoptosis. However, the onconase-induced activation of the caspase-9/caspase-3 cascade correlated with atypically little release of cytochrome c from mitochondria. In turn, the low levels of cytochrome c released from mitochondria correlated with a lack of detectable translocation of proapoptotic Bax from the cytosol onto mitochondria in response to onconase. This suggests the possibility of involvement of a different, potentially Bax- and cytochrome c-independent mechanism of caspase-9 activation in onconase-treated cells. As one possible mechanism, we demonstrate that procaspase-9 is released from mitochondria in onconase-treated cells. A detailed understanding of the molecular determinants of the cytotoxic action of onconase could provide means of positive or negative therapeutic modulation of the activity of this potent anticancer agent.

Thymidylate synthase activity and the cell growth are inhibited by the beta-carboline-benzoquinolizidine alkaloid deoxytubulosine.

Employing thymidylate synthase (TS) (5, 10-CH2-H4PteGlu: dUMP C-methyltransferase, EC 2.1.1.45), a key target enzyme in chemotherapy, the biological activity of the beta-carboline-benzoquinolizidine alkaloid deoxytubulosine (DTB) isolated from the Indian medicinal plant Alangium lamarckii has been evaluated and assessed for the first time. The TS employed in the present studies was purified from Lactobacillus leichmannii. The DTB was demonstrated to exhibit potent cytotoxicity and inhibited the cell growth of L. leichmannii, and DTB potently inhibited TS activity (IC50 = 40 microM). The DTB concentrations > 80 microM resulted in a total loss of the TS activity, thus suggesting that the beta-carboline-benzoquinolizidine alkaloid is a promising potential antitumor agent. The DTB binding to TS appears to be irreversible and tight through a possible covalent linkage. Although DTB strongly binds to DNA, it is not known whether DTB binds to RNA associated with TS. Inhibition kinetics showed that TS has a Ki value of 7 x 10(-6) M for DTB and that the inhibition is a simple linear "noncompetitive" type.

Mechanism of sodium channel blockade in the cardiotoxic action of emetine dihydrochloride in isolated cardiac preparations and ventricular myocytes of guinea pigs.

Emetine is used in the therapy of special forms of amebiasis and is abused as syrup of ipecac by persons with bulimia. Severe cardiac side effects were reported. Thus the intracellular microelectrode technique and the patch-clamp technique in the cell-attached mode were used to study the effects of emetine on the action potential and upstroke velocity (Vmax) in papillary muscles and Purkinje fibers of guinea pigs as well as on macroscopic and (S)-DPI 201-106-modified and unmodified single-sodium-channel current (I(Na)) of guinea-pig ventricular myocytes. Emetine caused a tonic block of Vmax and reduced I(Na) independent of frequency. Hill plots were linear, with slopes ranging from 0.96 to 1.06, suggestive of a first-order reaction. The current-voltage relation was not influenced, indicating a voltage-independent blockade of the sodium channels. The most prominent effects were an increase of sweeps without activity, a decrease of the fast component of the open-time distribution, an increase of the slow component of the closed-time distribution, and a reduction in the number of bursts per record. The amplitude of the unitary current was not changed. From the results, we conclude that I(Na) blockade contributes to the cardiotoxicity of emetine.

Competition of hydrophobic peptides, cytotoxic drugs, and chemosensitizers on a common P-glycoprotein pharmacophore as revealed by its ATPase activity.

The aim of the present study was to demonstrate that the modulation of P-glycoprotein (Pgp) ATPase activity by peptides, drugs, and chemosensitizers takes place on a common drug pharmacophore. To this end, a highly emetine-resistant Chinese hamster ovary cell line was established, in which Pgp constituted 18% of plasma membrane protein. Reconstituted proteoliposomes, the Pgp content of which was up to 40%, displayed a basal activity of 2.6 +/- 0.45 micromol of Pi/min/mg of protein, suggesting the presence of an endogenous Pgp substrate. This basal ATPase activity was stimulated (up to 5.2 micromol of Pi/min/mg of protein) by valinomycin and various Pgp substrates, whereas, to our surprise, gramicidin D, an established Pgp substrate, was inhibitory. Taking advantage of this novel inhibition of Pgp ATPase activity by gramicidin D, a drug competition assay was devised in which gramicidin D-inhibited Pgp ATPase was coincubated with increasing concentrations of various substrates that stimulate its ATPase activity. Gramicidin D inhibition of Pgp ATPase was reversed by Pgp substrates, including various cytotoxic agents and chemosensitizers. The inhibition of the basal ATPase activity and the reversal of gramicidin D inhibition of Pgp ATPase by its various substrates conformed to classical Michaelis-Menten competition. This competition involved an endogenous substrate, the inhibitory drug gramicidin D, and a stimulatory substrate. We conclude that the various MDR type substrates and chemosensitizers compete on a common drug binding site present in Pgp.

Functional reconstitution of P-glycoprotein reveals an apparent near stoichiometric drug transport to ATP hydrolysis.

We have recently described an ATP-driven, valinomycin-dependent 86Rb+uptake into proteoliposomes reconstituted with mammalian P-glycoprotein (Eytan, G. D., Borgnia, M. J., Regev, R., and Assaraf, Y. G. (1994) J. Biol. Chem. 269, 26058-26065). P-glycoprotein mediated the ATP-dependent uptake of 86Rb+-ionophore complex into the proteoliposomes, where the radioactive cation was accumulated, thus, circumventing the obstacle posed by the hydrophobicity of P-glycoprotein substrates in transport studies. Taking advantage of this assay and of the high levels of P-glycoprotein expression in multidrug-resistant Chinese hamster ovary cells, we measured simultaneously both the ATPase and transport activities of P-glycoprotein under identical conditions and observed 0.5-0.8 ionophore molecules transported/ATP molecule hydrolyzed. The amount of 86Rb+ ions transported within 1 min via the ATP- and valinomycin-dependent P-glycoprotein was equivalent to an intravesicular cation concentration of 8 mM. Thus, this stoichiometry and transport capacity of P-glycoprotein resemble various ion-translocating ATPases, that handle millimolar substrate concentrations. This constitutes the first demonstration of comparable rates of P-glycoprotein-catalyzed substrate transport and ATP hydrolysis.

Cytotoxicity of polyamines to Amoeba proteus: role of polyamine oxidase.

It has been shown that oxidation of polyamines by polyamine oxidases can produce toxic compounds (H2O2, aldehydes, ammonia) and that the polyamine oxidase-polyamine system is implicated, in vitro, in the death of several parasites. Using Amoeba proteus as an in vitro model, we studied the cytotoxicity to these cells of spermine, spermidine, their acetyl derivatives, and their hypothetical precursors. Spermine and N1-acetylspermine were more toxic than emetine, an amoebicidal reference drug. Spermine presented a short-term toxicity, but a 48-h contact time was necessary for the high toxicity of spermidine. The uptake by Amoeba cells of the different polyamines tested was demonstrated. On the other hand, a high polyamine oxidase activity was identified in Amoeba proteus crude extract. Spermine (theoretical 100%) and N1-acetylspermine (64%) were the best substrates at pH 9.5, while spermidine, its acetyl derivatives, and putrescine were very poorly oxidized by this enzyme (3-20%). Spermine oxidase activity was inhibited by phenylhydrazine (nil) and isoniazid (approximately 50%). Mepacrine did not inhibit the enzyme activity at pH 8. Neither monoamine nor diamine oxidase activity (approximately 10%) was found. It must be emphasized that spermine, the best enzyme substrate, is the most toxic polyamine. This finding suggests that knowledge of polyamine oxidase specificity can be used to modulate the cytotoxicity of polyamine derivatives. Amoeba proteus was revealed as a simple model for investigation of the connection between cytotoxicity and enzyme activity.

Cardiotoxicity of emetine dihydrochloride by calcium channel blockade in isolated preparations and ventricular myocytes of guinea-pig hearts.

1. The cardiotoxic effects of emetine dihydrochloride on mechanical and electrical activity were studied in isolated preparations (papillary muscles, sinoatrial and atrioventricular nodes, ventricular myocytes) of the guinea-pig heart. 2. Force of contraction was measured isometrically, action potentials and maximum rate of rise of the action potential were recorded by means of the intracellular microelectrode technique. Single channel L-type calcium current (Ba2+ ions as charge carrier) was studied with the patch-clamp technique in the cell-attached mode. 3. Emetine dihydrochloride (8-256 microM) reduced force of contraction in papillary muscles and spontaneous activity of sinoatrial and atrioventricular nodes concentration-dependently; the negative inotropic effect was abolished when the extracellular Ca2+ concentration was increased. 4. Maximum diastolic potential, action potential amplitude, maximum rate of rise of the action potential and the slope of the slow diastolic depolarization were decreased by emetine in sinoatrial as well as atrioventricular noes, while action potential duration was prolonged in both preparations (1-64 microM). 5. The amplitude of the L-type calcium single channel current was not altered by emetine dihydrochloride, while average open state probability was decreased concentration-dependently (10, 30 and 60 microM). 6. The most prominent effect of emetine dihydrochloride on single channel current was an increase of sweeps without activity. 7. At 60 microM, emetine dihydrochloride caused a decrease of the mean open time an increase of the mean closed time. The number of openings per record and number of bursts per record were reduced. 8. It is concluded that emetine dihydrochloride produces an L-type calcium channel block which might contribute to its cardiac side effects.

Effects of pharmacological interventions on emetine cardiotoxicity in isolated perfused rat hearts.

The cardiotoxicity of emetine continues to be a significant clinical problem. The purpose of this study was to investigate the effect of several mechanistic interventions, including ICRF-187, an iron-chelating agent which protects against doxorubicin toxicity, atropine, and fructose-1,6-bisphosphate (FBP) on the toxicity of emetine in our isolated, perfused rat heart model. The model includes functional, electrocardiographic, and biochemical determinations in the same preparation. Atropine and ICRF-187 had no effect on the time needed for emetine to induce ventricular asystole, while FBP significantly increased this time. Administration of 47 microM atropine, 300 microM FBP, or 1 mM FBP decreased the release of lactate dehydrogenase (LDH) into the coronary effluent, while ICRF-187 had no effect. These pharmacological interventions variably changed the amplitude of the biphasic response of the coronary flow to emetine. Finally, FBP was very effective in slowing the rate of QRS-waveform degeneration in the perfused hearts. Emetine caused PR- and QRS-prolongation which was not altered by FBP.

The CRY1 gene in Chlamydomonas reinhardtii: structure and use as a dominant selectable marker for nuclear transformation.

We have cloned and sequenced the CRY1 gene, encoding ribosomal protein S14 in Chlamydomonas reinhardtii, and found that it is highly similar to S14/rp59 proteins from other organisms, including mammals, Drosophila melanogaster, and Saccharomyces cerevisiae. We isolated a mutant strain resistant to the eukaryotic translational inhibitors cryptopleurine and emetine in which the resistance was due to a missense mutation (CRY1-1) in the CRY1 gene; resistance was dominant in heterozygous stable diploids. Cotransformation experiments using the CRY1-1 gene and the gene for nitrate reductase (NIT1) produced a low level of resistance to cryptopleurine and emetine. Resistance levels were increased when the CRY1-1 gene was placed under the control of a constitutive promoter from the ribulose bisphosphate carboxylase/oxygenase small subunit 2 (RBCS2) gene. We also found that the 5' untranslated region of the CRY1 gene was required for expression of the CRY1-1 transgene. Direct selection of emetine-resistant transformants was possible when transformed cells were first induced to differentiate into gametes by nitrogen starvation and then allowed to dedifferentiate back to vegetative cells before emetine selection was applied. With this transformation protocol, the RBCS2/CRY1-1 dominant selectable marker gene is a powerful tool for many molecular genetic applications in C. reinhardtii.

Experimental emetine myopathy: enzyme histochemical, electron microscopic, and immunomorphological studies.

Ipecac, containing emetine hydrochloride, is used by patients with anorexia nervosa to induce vomiting. Its chronic usage may result in a myopathy and a cardiomyopathy, the former marked by cytoplasmic bodies. We studied myopathological changes after daily injections of female Wistar rats with emetine hydrochloride intraperitoneally for periods of 4, 5, 9, and 10 weeks. The extensor digitorum longus muscle and the soleus muscle showed core-like lesions, streaming of the z-discs, nemaline bodies, cytoplasmic bodies, and spheroid cytoplasmic bodies. Immunomorphological studies revealed increased amounts of desmin. During a period of repair, i.e., 2, 4, and 6 weeks after termination of emetine application, these myopathological alterations faded while proliferation of the T-tubular system, i.e., honeycomb structures, was more often prevalent. Pathological features completely disappeared between 6 and 12 weeks of recovery.

Continued development and unconditioned stimulus characterization of selectively bred lines of taste aversion prone and resistant rats.

This report updates the bidirectional selective breeding of taste aversion (TA) prone (TAP) and TA resistant (TAR) rat lines from the 8th through the 22nd generations. A palatable saccharin solution and the aversive consequences of a cyclophosphamide injection are the respective conditioned stimulus (CS) and unconditioned stimulus (US) of line development. Nonsibling matings within each of the two extremes of TA conditionability have produced TAP and TAR lines having markedly different TA propensities. As previously reported, the substitution of a rotational (i.e., motion sickness) US for cyclophosphamide during TA conditioning also produced characteristic line differences in conditioned taste aversion acquisition. The present report extends the effective line separating USs to include injections of lithium chloride, emetine hydrochloride, and EtOH. A range of EtOH dose levels produced dose-dependent TAs within TAP rats but failed to induce TAs in TAR rats. Following the conclusion of TA testing, the administration of a hypnotic EtOH dose produced equivalent loss of righting capability and equivalent hypothermia in both TAP and TAR rats. The line differences in EtOH induced TA conditionability therefore do not reflect general line differences in EtOH sensitivity. The lines may be useful within studies of biological bases of TA conditionability and animal analog studies of prevention and treatment of alcohol dependence.

Reversible emetine-induced myopathy with ECG abnormalities: a toxic myopathy.

A young anorexic woman is described with a history of progressive muscle weakness following chronic ingestion of syrup of ipecac that was used in an attempt at weight control. Electrocardiogram (ECG) showed T-wave inversion in all leads and prolongation of the Q-T interval. Electromyography was abnormal. Muscle biopsy revealed a randomized, generalized, predominantly type-2 fibre atrophy and structural alterations in oxidative enzyme stains, such as targetoid and moth-eaten fibres. A few necrotic cells were actively phagocytosed. Electron microscopy revealed severe sarcomeric abnormalities with Z-line streaming, myofibrillar disorganization and increased lysosomal activity. After discontinuation of ipecac syrup, the patient noted a gradual improvement in her condition. The ECG became normal. Emetine and related drug-induced myopathies are uncommon but are of experimental interest because of their effects on mitochondria and the light they shed on autophagic mechanisms in muscle.

Detection of cardiotoxic hazards.

For the detection and quantitative assessment of cardiovascular toxicity, the morphological and biochemical studies used in routine toxicity tests must be supplemented with functional and metabolic measurements. Examples are given which show that the cardiotoxic effects of allylamine and emetine can be detected early by blood pressure and ECG monitoring. The same techniques are helpful in assessing potential drug interactions. For the hypoglycemic agent MTG measurement of mitochondrial function proved to be the most adequate method to demonstrate cardiotoxic hazard.

A model system for measuring comparative toxicities of cardiotoxic drugs with cultured rat heart myocytes, endothelial cells and fibroblasts. I. Emetine, chloroquine and metronidazole.

Neonatal rat hearts were separated into separate cultures of beating myocytes (M cells), endothelial cells (E cells) and fibroblasts (F cells). Their susceptibilities to the toxic effects of emetine, chloroquine and metronidazole were then compared using a quantitative metabolic inhibition test (QMIT) and morphologic and beating changes as indices of injury. Measurements on the same cultures were made at 6 h and 12 h daily for 7 days with E and F cells; with M cells for 3 days. Metronidazole was non-toxic for all cell types at 810 micrograms/ml, whether as the parent compound or after attempted rat liver microsomal activation. QMIT data, integrated as time-concentration effects, showed all cell responses to either emetine or chloroquine to be parallel (P less than 0.05), and their order of susceptibility to be: E greater than M greater than F cells. Although morphologic signs of injury and changes in beating are not readily evaluated statistically, there was a general parallelism between these indices of injury and those of QMIT. As judged by QMIT emetine was more toxic than chloroquine. However, at equivalent QMIT grades chloroquine produced greater effects on morphology and beating. Toxic concentration-50 values for chloroquine with the QMIT were similar to reported human toxic and lethal blood concentrations.

Remodeling of sperm chromatin following fertilization: nucleosome repeat length and histone variant transitions in the absence of DNA synthesis.

Within the first cell cycle following fertilization the average nucleosomal repeat length of sea urchin male pronuclear chromatin declines by 30-40 base pairs to a value typical of that found in the embryo. This decline occurs after a lag of about 30 min postfertilization, and is accompanied by replication of the male chromatin and accumulation of cleavage-stage (CS) core histone variants. When replication is inhibited by greater than 95% with aphidicolin, the decline in repeat length still occurs, although it is slightly retarded. The decline in repeat length also occurs when protein synthesis is blocked by greater than 98% and DNA synthesis by 60-70% with emetine. The adjustment of nucleosome repeat length therefore can occur in vivo without extensive movement of replication forks across the length of the chromatin, or normal progression of the cell cycle, and appears to require no proteins synthesized postfertilization. Blocking of DNA synthesis or protein synthesis also does not prevent the normal histone variant transitions involved in male pronuclear chromatin remodeling. Although their accumulation is slowed, CS core variants eventually become the predominant male pronuclear histones in their classes when replication is inhibited. Since a shortening of the average nucleosomal repeat length of approximately 10-20% is not sufficient to account for this large acquisition of CS variants, some of the sperm (Sp) core histones are probably displaced from the replication-blocked pronucleus. Therefore, accumulation of CS H2A and CS H2B are temporally correlated with the repeat length transition, whereas replication, normal progression of the cell cycle, and the early histone transitions involving SpH1 and SpH2B are not.