Critical Dose Range Research Articles

Two-miRNA-based finger-stick assay for estimation of absorbed ionizing radiation dose.

Nuclear radiation and radioactive fallouts resulting from a nuclear weapon detonation or reactor accidents could result in injuries affecting multiple sensitive organs, defined as acute radiation syndrome (ARS). Rapid and early estimation of injuries to sensitive organs using markers of radiation response is critical for identifying individuals who could potentially exhibit ARS; however, there are currently no biodosimetry assays approved for human use. We developed a sensitive microRNA (miRNA)-based blood test for radiation dose reconstruction with ±0.5 Gy resolution at critical dose range. Radiation dose-dependent changes in miR-150-5p in blood were internally normalized by a miRNA, miR-23a-3p, that was nonresponsive to radiation. miR-23a-3p was not highly expressed in blood cells but was abundant in circulation and was released primarily from the lung. Our assay showed the capability for dose estimation within hours to 1 week after exposure using a drop of blood from mice. We tested this biodosimetry assay for estimation of absorbed ionizing radiation dose in mice of varying ages and after exposure to both improvised nuclear device (IND)-spectrum neutrons and gamma rays. Leukemia specimens from patients exposed to fractionated radiation showed depletion of miR-150-5p in blood. We bridged the exposure of these patients to fractionated radiation by comparing responses after fractionated versus single acute exposure in mice. Although validation in nonhuman primates is needed, this proof-of-concept study suggests the potential utility of this assay in radiation disaster management and clinical applications.

The Cannabinoid CB1 Antagonist TM38837 With Limited Penetrance to the Brain Shows Reduced Fear-Promoting Effects in Mice.

Rimonabant was the first selective CB1 antagonist/inverse agonist introduced into clinical practice to treat obesity and metabolic-related disorders. It was withdrawn from market due to the notably increased rates of psychiatric side effects. We have evaluated TM38837, a novel, largely peripherally restricted CB1 antagonist, in terms of fear-promoting consequences of systemic vs. intracerebral injections. Different groups of male C57BL/6 N mice underwent auditory fear conditioning, followed by re-exposure to the tone. Mice were treated per os (p.o.) with TM38837 (10, 30, or 100 mg/kg), rimonabant (10 mg/kg; a brain penetrating CB1 antagonist/inverse agonist which served as a positive control), or vehicle, 2 h prior the tone presentation. Only the high dose of TM38837 (100 mg/kg) induced a significant increase in freezing behavior, similar to that induced by rimonabant (10 mg/kg) (p < 0.001). If injected into the brain both TM38837 (10 or 30 μg/mouse) and rimonabant (1 or 10 μg/mouse) caused a sustained fear response to the tone, which was more pronounced after rimonabant treatment. Taken together, TM38837 was at least one order of magnitude less effective in promoting fear responses than rimonabant. Given the equipotency of the two CB1 antagonists with regard to weight loss and metabolic syndrome-like symptoms in rodent obesity models, our results point to a critical dose range in which TM3887 might be beneficial for indications such as obesity and metabolic disorders with limited risk of fear-promoting effects.

From the Cover: Manganese Stimulates Mitochondrial H2O2 Production in SH-SY5Y Human Neuroblastoma Cells Over Physiologic as well as Toxicologic Range.

Manganese (Mn) is an abundant redox-active metal with well-characterized mitochondrial accumulation and neurotoxicity due to excessive exposures. Mn is also an essential co-factor for the mitochondrial antioxidant protein, superoxide dismutase-2 (SOD2), and the range for adequate intake established by the Institute of Medicine Food and Nutrition Board is 20% of the interim guidance value for toxicity by the Agency for Toxic Substances and Disease Registry, leaving little margin for safety. To study toxic mechanisms over this critical dose range, we treated human neuroblastoma SH-SY5Y cells with a series of MnCl2 concentrations (from 0 to 100 μM) and measured cellular content to compare to human brain Mn content. Concentrations ≤10 μM gave cellular concentrations comparable to literature values for normal human brain, whereas concentrations ≥50 μM resulted in values comparable to brains from individuals with toxic Mn exposures. Cellular oxygen consumption rate increased as a function of Mn up to 10 μM and decreased with Mn dose ≥50 μM. Over this range, Mn had no effect on superoxide production as measured by aconitase activity or MitoSOX but increased H2O2 production as measured by MitoPY1. Consistent with increased production of H2O2, SOD2 activity, and steady-state oxidation of total thiol increased with increasing Mn. These findings have important implications for Mn toxicity by re-directing attention from superoxide anion radical to H2O2-dependent mechanisms and to investigation over the entire physiologic range to toxicologic range. Additionally, the results show that controlled Mn exposure provides a useful cell manipulation for toxicological studies of mitochondrial H2O2 signaling.

Different biocompatibility of crystalline triamcinolone deposits on retinal cells in vitro and in vivo

Epiretinal deposits of triamcinolone acetonide (TA) can be detrimental to retinal cells in vitro as several laboratory studies have shown. This contrasts with the good clinical experience of intravitreal TA use. We investigated the effect of TA crystals on retinal cells concerning the critical dose range, a potential cell recovery, the drug–tissue interaction and what protective biological factors could explain the discrepancy between in vivo and in vitro results. A human retinal pigment epithelium cell line (ARPE19) and transformed rat retinal ganglion cells (RGC5) were used. Purified TA crystals were either added directly on top of the cell cultures or on top of membrane filter inserts, basement membrane sheets or porcine vitreous with the cells growing underneath. To determine the number of live versus dead cells fluorescent stains were used. Proliferation and viability were measured using the MTT assay and the mean inhibitory dose (ID 50) calculated with or without a filter. Cell recovery was measured after transient TA exposure (0.01–1 mg/ml) compared to continuous exposure after 7 days. To exclude a mere mechanical effect of epicellular deposits the TA crystals were replaced by glass pearls in a serum-free medium and the MTT toxicity assay was performed after 24 h. Without direct contact of TA crystals with the cells only a moderate decrease of mitochondrial activity was observed that fully recovered after transient exposure and showed a clinically safe ID 50 of 7.7 mg/ml. In contrast, direct exposure to even minute crystalline deposits for 7 days caused a rapid progressive and irreversible cell death being significant far below clinically used concentrations (ID 50 0.058 mg/ml). Direct exposure to glass pearls did not show any loss of viability. Both basement membrane sheets and vitreous reliably prevented direct cytotoxicity to underlying retinal ganglion cells. Our findings suggest that irreversible TA cytotoxicity in a cell culture setting occurs earlier than previously assumed in the presence of even minute epicellular deposits. But in most clinical situations epiretinal TA deposits seem not to be harmful to ocular cells as protective biological factors may prevent close apposition of TA crystals to susceptible retinal cells. However, in eyes that have undergone vitrectomy with ILM peeling epimacular deposits could be critical.

Health Implications of Drug Interactions

The awareness of the digoxin-quinidine interaction in the mid-70s led to increased interest in drug interactions. Much has been published on the subject in the form of interaction reports, handbooks, lists, cards, discs etc. However, only a few interactions in the literature have clinical significance and can be remembered by thinking in terms of groups, pharmacokinetics and probabilities. It is important to realise that drugs in a given class will have similar properties. Thus, phenylbutazone, like any other nonsteroidal anti-inflammatory drug (NSAID), is likely to potentiate the effects of warfarin. A knowledge of drug pharmacokinetics is also essential; a highly protein-bound drug may displace another when they are administered together. Remembering those drugs which induce liver enzyme formation will prevent their co-administration with drugs which have a critical dose-range. If a general practitioner can remember the few drugs in clinical practice with a narrow therapeutic index, he can consult a handbook before anything else is prescribed. Some interactions will rarely be encountered in daily general practice, i.e. those associated with tuberculosis treatment or anesthetic agents. Other interactions, e.g. the interaction between antihypertensive treatment and over-the-counter medication containing phenylpropanolamine, are more common. While most drug interactions can be avoided by thinking in terms of groups, pharmacokinetics and probabilities, some learning by rote is required, e.g. the potential for heart failure with concomitant beta-blocker and nifedipine therapy. In general, a schematic approach using thinking in terms of groups, pharmacokinetics and probabilities will prevent most clinically significant drug interactions; the rest can be avoided by consulting appropriate handbooks and specialists.

Effects of methyl β-carboline-3-carboxylate, Ro 15-1788 and CGS 8216 on muscle tone in genetically spastic rats

Genetically spastic rats were used for studying the effect on muscle tone of β-carboline-3-carboxylic acid methylester (β-CCM), an inverse benzodiazepine (BDZ) agonist, and that of Ro 15-1788 and CGS 8216, both putative antagonists of pharmacological actions of BDZs. These animals exhibit pathologically increased muscle tone, which can be recorded and quantified in the electromyogram (EMG) of the gastrocnemius (GS) muscle. β-CCM, 2.5 and 3.0 mg/kg i.p., augmented the tonic activity in the EMG of GS muscle in spastic rats while it did not modify muscle tone at doses of 1.0 and 2.0 mg/kg. Diazepam, 0.4 mg/kg i.p., and Ro 15-1788, 5 mg/kg i.p., but not CGS 8216, 5 mg/kg i.p., antagonised the effect of the β-carboline on muscle tone. Ro 15-1788, at doses of 0.1–5 mg/kg i.p., did not affect muscle tone in the genetically spastic rats, whilst at doses of 25–200 mg/kg the imidazodiazepine dose dependently increased the tonic activity in the EMG. The action of Ro 15-1788, 50 mg/kg i.p., was reversed by diazepam, 0.4 mg/kg i.p., and β-CCM, 2 mg/kg i.p., while CGS 8216, 5 mg/kg i.p., facilitated the effect of Ro 15-1788 on muscle tone. CGS 8216 at doses of 5 and 25 mg/kg i.p. was devoid of any effect on muscle tone, whilst at doses of 50 and 200 mg/kg the pyrazoloquinoline increased the tonic activity recorded in the EMG from the GS muscle of the spastic rats. The effect of CGS 8216, 50 mg/kg i.p., on muscle tone was blocked by diazepam, 0.4 mg/kg i.p., while Ro 15-1788, 5 mg/kg i.p., and β-CCM, 2 mg/kg i.p., augmented the action of CGS 8216. Alone, neither diazepam, 0.4 mg/kg, nor β-CCM, 2.0 mg/kg influenced the activity in the EMG of genetically spastic rats. This study demonstrates that an increase in muscle tone can be achieved via an inverse agonist action at BDZ receptors. The results indicate that BDZ receptors are involved in the effect of β-CCM and Ro 15-1788 on muscle tone and suggest that at a critical dose range Ro 15-1788 possesses inverse agonist activity at BDZ receptors in vivo. The failure of both β-CCM and Ro 15-1788 to prevent the augmenting effect of CGS 8216 on muscle tone might indicate a different mechanism of interaction of the pyrazoloquinoline at the BDZ receptor.

Involvement of 5-HT2 receptors in the LSD- and L-5-HTP-induced suppression of lordotic behavior in the female rat.

Copulatory behavior in the ovariectomized rat, the lordotic response (L.R.), was induced by estrogen followed by progesterone. L.R. is inhibited by lysergic acid diethylamide (LSD) (greater than or equal to 0.05 mg/kg) and by Levo-5-hydroxytryptophan (L-5-HTP) (greater than or equal to 2.5 mg/kg). The effects of the putative 5-HT antagonists lisuride, metergoline, methysergide, mianserin, cinanserin, cyproheptadine, pirenperone and altanserin on the LSD-induced inhibition of L.R. were tested. Lisuride, metergoline, methysergide and mianserin were found to have no LSD-blocking effect. In contrast, cinanserin, cyproheptadine and pirenperone acted antagonistically to LSD, within a critical dose range. The selective 5-hydroxytryptamine2 (5-HT2) receptor antagonist altanserin effectively prevented the LSD-induced inhibition of L.R., and the doses required (0.05-0.20 mg/kg) indicated a comparatively high antagonistic potency. In addition altanserin (0.2 mg/kg) effectively prevented the lordosis inhibitory effect induced by L-5-HTP (2.5 mg/kg), after pretreatment with pargyline and RO4-4602. It is suggested that the suppression of copulatory behavior caused by LSD and L-5-HTP is mediated by 5-HT2 receptors.

Conditioned taste aversion induced by 2,4,5-trichlorophenoxyacetic acid: Dose-response and preexposure effects

Taste aversion to a.1% saccharin (sacc) solution was induced in rats by pairing sacc consumption with an oral dose of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). In Experiment 1, a critical dose range for producing taste aversion with 2,4,5-T was found from between 15 and 40 mg/kg and upwards. In Experiment 2, preconditioning exposure of the animals to sacc and/or the 2,4,5-T injection reduced the strength of the aversion. The possible risk of producing alterations of food preferences in wildlife through a widespread use of 2,4,5-T as a herbicide is discussed.

Ion contents of human lymphocytes: The effects of concanavalin A and ouabain

It has been suggested that mitogens may activate the Na,K-ATPase to cause an increased cell K + which may then trigger metabolic events initiating DNA synthesis. To test this hypothesis, human lymphocytes were treated with ouabain and concanavalin A (ConA) and their ion contents measured directly by atomic absorption. Ouabain decreased cell K + with a critical dose range, 10 −8 to 10 −7 M, similar to that which inhibits mitogenesis, and induced a mole-for-mole replacement of K + by Na +. ConA, in non-toxic, mitogenic doses, also caused a rapid and a sustained decrease in cell K +, but, unlike ouabain, did not induce replacement of the lost K + by Na +, and the total K + + Na + was reduced in spite of a normal water content. Thus, although normal Na,K-ATPase function may be required for mitogenesis, ConA does not affect cell K + and Na + simply by activating the Na,K-ATPase. Stable-state K + and Na + contents were then determined over a wide range of external K + levels, and analyzed by the equivalent of a Hill plot. The normal cooperative uptake of K + was inhibited in an allosteric manner by ouabain, while ConA failed to alter significantly the critical parameters of K +-Na + exchange and the cooperativity in K + uptake. We suggest that K + is not a specific trigger in the initiation of mitogenesis, but that changes in K + flux and content reflect a change within the physical state of an underlying macromolecular assembly that is poised to respond to the mitogenic stimulus in a critical cooperative fashion.