Nicotine and Cytisine Embryotoxicity in the Experimental Zebrafish Model.

Tobacco smoking is one of the most serious health problems. Potentially lethal effects of nicotine for adults can occur with as little as 30 to 60 mg, although severe symptoms can arise with lower doses. Furthermore, the route of administration also influences the toxicity. Cytisine is one of the most popular medications in nicotinism treatment. Like nicotine, cytisine is a plant alkaloid, signaling through nicotinic acetylcholine receptors. Our study evaluated the effects of cytisine in nicotine-induced embryotoxic effects using zebrafish larvae. We examined the teratogenicity of nicotine and cytisine alone or in combination. Nicotine increased mortality and delayed hatching of zebrafish larvae in a dose-dependent manner. Cytisine did not affect mortality in a wide range of concentrations, and hatching delay was observed only at the highest concentrations, above 2 mM. Administering compounds together partially reduced the adverse teratogenic effect induced by nicotine alone. The protective effect of cytisine against the nicotine effect, observed in zebrafish, will contribute to future studies or treatments related to nicotine addiction or prenatal nicotine exposure in humans.

Cytisine and cytisine derivatives. More than smoking cessation aids.

Cytisine, a natural bioactive compound that is mainly isolated from plants of the Leguminosae family (especially the seeds of Laburnum anagyroides), has been marketed in central and eastern Europe as an aid in the clinical management of smoking cessation for more than 50 years. Its main targets are neuronal nicotinic acetylcholine receptors (nAChRs), and pre-clinical studies have shown that its interactions with various nAChR subtypes located in different areas of the central and peripheral nervous systems are neuroprotective, have a wide range of biological effects on nicotine and alcohol addiction, regulate mood, food intake and motor activity, and influence the autonomic and cardiovascular systems. Its relatively rigid conformation makes it an attractive template for research of new derivatives. Recent studies of structurally modified cytisine have led to the development of new compounds and for some of them the biological activities are mediated by still unidentified targets other than nAChRs, whose mechanisms of action are still being investigated. The aim of this review is to describe and discuss: 1) the most recent pre-clinical results obtained with cytisine in the fields of neurological and non-neurological diseases; 2) the effects and possible mechanisms of action of the most recent cytisine derivatives; and 3) the main areas warranting further research.

Cytisine inhibits the protective activity of various classical and novel antiepileptic drugs against 6 Hz-induced psychomotor seizures in mice.

BACKGROUND: Cytisine (CYT) is a partial agonist of brain α4ß2 nicotinic acetylcholine receptors widely used in Central/Eastern Europe for smoking cessation. OBJECTIVES: This study evaluated the effect of CYT on the ability of classical and novel antiepileptic drugs to prevent seizures evoked by the 6-Hz test, a model of psychomotor seizures in mice thought as a model of drug-resistant seizures. RESULTS: CYT administered intraperitoneally (i.p.) in a dose of 2 mg kg-1 significantly inhibited the anticonvulsant activity of lacosamide, levetiracetam, and pregabalin, increasing their median effective doses 50 (ED50) values from 6.88 to 10.52 mg kg-1 (P < 0.05) for lacosamide, from 22.08 to 38.26 mg kg-1 (P < 0.05) for levetiracetam, and from 40.48 to 64.61 mg kg-1 (P < 0.01) for pregabalin, respectively. There were no significant changes in total brain concentrations of lacosamide, levetiracetam, and pregabalin following CYT i.p. administration. CYT administered in a dose of 2 mg kg-1 failed to change the protective action of clobazam, clonazepam, phenobarbital, tiagabine, and valproate in the 6-Hz test. Neither CYT (2 mg kg-1) alone nor its combination with the anticonvulsant drugs (at their ED50 values from the 6-Hz test) affected motor coordination; skeletal muscular strength and long-term memory, as determined in the chimney; and grip strength and passive avoidance tests, respectively. CONCLUSION: CYT-evoked alterations in the protection provided by some antiepileptic drugs against seizures can be of serious concern for epileptic smokers, who might demonstrate therapeutic failure to lacosamide, levetiracetam, and pregabalin, resulting in possible breakthrough seizure attacks.

Polyamine modification by acrolein exclusively produces 1,5-diazacyclooctanes: a previously unrecognized mechanism for acrolein-mediated oxidative stress.

Acrolein, a toxic unsaturated aldehyde generated as a result of oxidative stress, readily reacts with a variety of nucleophilic biomolecules. Polyamines, which produced acrolein in the presence of amine oxidase, were then found to react with acrolein to produce 1,5-diazacyclooctane, a previously unrecognized but significant downstream product of oxidative stress. Although diazacyclooctane formation effectively neutralized acrolein toxicity, the diazacyclooctane hydrogel produced through a sequential diazacyclooctane polymerization reaction was highly cytotoxic. This study suggests that diazacyclooctane formation is involved in the mechanism underlying acrolein-mediated oxidative stress.

Synergetic toxic effect of an explosive material mixture in soil.

Explosives materials are stable in soil and recalcitrant to biodegradation. Different authors report that TNT (2,4,6-trinitrotoluene), RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) and HMX (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine) are toxic, but most investigations have been performed in artificial soil with individual substances. The aim of the presented research was to assess the toxicity of forest soil contaminated with these substances both individually as well in combinations of these substances. TNT was the most toxic substance. Although RDX and HMX did not have adverse effects on plants, these compounds did cause earthworm mortality, which has not been reported in earlier research. Synergistic effects of explosives mixture were observed.

Primary constituents of blue cohosh: quantification in dietary supplements and potential for toxicity.

Dietary supplements containing dried roots or extracts of the roots and/or rhizomes of blue cohosh (Caulophyllum thalictroides) are widely available. This botanical has a long history of use by Native Americans and its use continues to the present day. The primary constituents of blue cohosh are its alkaloids and saponins. The structures of the alkaloids magnoflorine, baptifoline, anagyrine, and N-methylcytisine have been known for many years. The last 10 years have seen a great increase in isolation and identification of the large number of saponins present in blue cohosh. Important developments in nuclear magnetic resonance techniques have contributed substantially to the increase in elucidation of the structures of the complex saponins. Several authors have described quantitative methods for both the alkaloids and saponins in blue cohosh. Such methods have made it possible to quantify these constituents in dietary supplements containing this botanical ingredient. Concentrations of both alkaloids and saponins vary substantially in dietary supplements of blue cohosh. The nicotinic alkaloid, N-methylcytisine, a potent toxicant, has been found in all dietary supplements of blue cohosh analyzed. The teratogenic alkaloid anagyrine has been found in some but not all dietary supplements.

Accumulation and effects of octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) exposure in the green anole (Anolis carolinensis).

Environmental contamination by energetic compounds is an increasing international concern, although little is known of their accumulation in and affect on wildlife. Reptiles are often good models for contaminants studies due to natural history traits that increase their potential for exposure. We report a study to assess accumulation and effects of octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX, High Melting Explosive) in green anoles (Anolis carolinensis). Acute oral toxicity (LD(50)) was estimated to exceed 2000 mg/kg body weight in adult male and female anoles using a standard up-and-down method. Accumulation of HMX was assessed in adult females via dietary exposure and into eggs by two routes (directly from the soil and via maternal transfer). HMX readily accumulated into adult females in a dose-dependent manner and into eggs following both exposure pathways. However, total HMX in soil-exposed eggs was up to 40-times greater than those exposed via maternal transfer. Although there was a suggestion of an HMX-induced reduction in body weight in adult females, overall there were no effects observed over the 12 week exposure period. The only significant effect on eggs was a 50% reduction in hatching success for eggs exposed to 2000 mg/kg HMX in the soil during incubation. Growth and survival of hatchlings was not affected by HMX exposure. Our results demonstrate that HMX accumulates through the food chain and into eggs from the soil, but likely poses minimal threat to lizards except to hatching success in eggs incubated in soils with HMX levels near maximum environmental concentrations.

Cytotoxicity of new pyrazino[1,2-b]isoquinoline and 6,15-iminoisoquino[3,2-b]3-benzazocine compounds.

Looking for optimised analogues of compound 2 that might be useful in colon cancer therapy, we here explore the in vitro cytotoxicity against MDA-MB 231 human breast carcinoma, A-549 human lung carcinoma and HT-29 human colon carcinoma cell lines of several analogues and derivatives. The effect of the R2-substituent and/or the introduction of an arylmethyl side-chain at C-3, as well as the presence of a double bond in the skeleton or a methoxy group at C-1 have been investigated. New 6,15-iminoisoquino[3,2-b]3-benzazocine compounds, related to the saframycin family, in which the C(7)-N(8)-C(9)-substructure contains a lactam function, a fused oxazolidine or an aminonitrile function were also studied, and many of them showed low micromolar GI50 values.

Toxicity and bioconcentration evaluation of RDX and HMX using sheepshead minnows in water exposures.

Lethal effects of the explosives RDX and HMX were assessed using ten-day water exposures to juvenile sheepshead minnows (Cyprinodon variegatus). For RDX, maximum mortality occurred during the first two days of exposure with a 10-d median lethal concentration (LC50) of 9.9 mg L(-1). The RDX 10-d median lethal residue (LR50) was 9.6 mg kg(-1) (34.9 µmol kg(-1)) wet weight (ww), the first RDX critical body residue reported for fish. Previous investigations reported that RDX body residues in marine amphipods up to 96 µmol kg(-1) ww and in marine mussels up to 86 µmol kg(-1) ww failed to result in significant mortality. The highest HMX concentration tested, corresponding to its apparent solubility limit in seawater (2.0 mg L(-1)), and the associated mean body residue (3 mg kg(-1) or 14 µmol kg(-1) ww) resulted in no significant mortality for exposed minnows. The mean 10-d bioconcentration factors for RDX (0.6-0.9 L kg(-1)) and HMX (0.3-1.6 L kg(-1)) were typically lower than 1, reflecting the low bioaccumulative potential for these compounds.

Toxicity of octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) in three vertebrate species.

The explosive, octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine or high-melting explosive (HMX), has been found in soils in areas used for testing and training by the military. Many of these areas contain habitat for valued wildlife species. In an effort to better understand the environmental consequences from exposure, a reptilian (western fence lizard [Sceloporus occidentalis]), an amphibian (red-backed salamander [Plethodon cinereus]), and a mammalian species (rabbit [Oryctolagus cuniculus]) were exposed to HMX under controlled laboratory conditions. Lizards and rabbits were exposed to HMX by way of corn oil through gavage, and salamanders were exposed to HMX in soil. Two deaths occurred from acute oral exposures to lizards to 5000 mg HMX/kg BW. Histological and gross pathologic assessment suggested gut impaction as a possible cause of death. Salamanders exposed to concentrations of HMX in soil < or = 1970 mg HMX/kg soil for 10 days did not show adverse effects. Rabbits, however, showed neurologic effects manifested as hyperkinetic events with convulsions at > 24 h after oral exposures. An LD(50) for rabbits was calculated as 93 mg/kg (95% confidence interval 76-117). A subacute 14-day testing regime found a lowest observed effect level of 10 mg/kg-d and a no observed adverse effect level of 5 mg/kg-d based on hyperkinesia and seizure incidence, although changes suggesting functional hepatic alterations were also found. These data suggest that physiologic differences between species, particularly in gastrointestinal structure and function, can affect the absorption of HMX and hence lead to marked differences in toxicity from exposure to the same compound.

The effect of body condition on serum concentrations of two teratogenic alkaloids (anagyrine and ammodendrine) from lupines (Lupinus species) that cause crooked calf disease.

Several species of lupine (Lupinus spp.) are toxic to livestock, causing death losses in sheep and cattle but more commonly crooked calf disease in pregnant range cows. The major toxic alkaloids in lupine are of the quinolizidine alkaloid group and include the teratogen anagyrine, which is primarily responsible for crooked calf disease. Lupines also contain teratogenic piperidine alkaloids including ammodendrine. Previous work in sheep has shown that lupine alkaloid clearance may be influenced by the animal's physiological status. Therefore, the purpose of this study was to determine if differences in body condition of cattle would alter the absorption and elimination of anagyrine or ammodendrine given in a single oral dose as Lupinus leucophyllus or Lupinus sulphureus, respectively. Mature non-lactating cows in low body condition (LBC, n = 4) and high body condition (HBC, n = 4) received a single dose of dry ground lupine plant (2.0 g/kg of BW) via oral gavage. Lupinus leucophyllus (anagyrine) was dosed first; then after 21 d the same animals were dosed with L. sulphureus (ammodendrine). Blood samples were taken via jugular venipuncture 0 to 60 h after dosing. Serum anagyrine and ammodendrine concentrations were evaluated. The concentration of anagyrine was greater (P = 0.001) in the HBC group and peaked 2 h after dosing versus 12 h in LBC cows. Similarly for ammodendrine, the alkaloid concentration peaked at 3 h after dosing for the HBC group compared with 6 h for the LBC group (P = 0.001). Area under the curve tended to differ (P

Toxicity and uptake of cyclic nitramine explosives in ryegrass Lolium perenne.

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX), and 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane (CL-20) are cyclic nitramines used as explosives. Their ecotoxicities have been characterized incompletely and little is known about their accumulation potential in soil organisms. We assessed the toxicity and uptake of these explosives in perennial ryegrass Lolium perenne L. exposed in a Sassafras sandy loam (SSL) or in a sandy soil (DRDC, CL-20 only) containing contrasting clay contents (11% and 0.3%, respectively). A 21-d exposure to RDX, HMX or CL-20 in either soil had no adverse effects on ryegrass growth. RDX and HMX were translocated to ryegrass shoots, with bioconcentration factors (BCF) of up to 15 and 11, respectively. In contrast, CL-20 was taken up by the roots (BCF up to 19) with no translocation to the shoots. These studies showed that RDX, HMX, and CL-20 can accumulate in plants and may potentially pose a risk of biomagnification across the food chain.

Effects of HMX exposure upon metabolic rate of northern bobwhite quail (Colinus virginianus) in ovo.

We evaluated the use of the gas exchange rate as an ecologically relevant indicator of chemical stress in avian embryos/eggs. Northern bobwhite quail (Colinus virginianus) were exposed to octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) via feed containing nominal concentrations of 0, 12.5, 50.0, and 125.0 mg kg(-1). Metabolic rates (oxygen consumption) of developing quail eggs were then measured via respirometry to examine potential effects of HMX exposure. Metabolic rates were examined on 5, 9, and 21 d of incubation. Next, concentrations of HMX in embryos/eggs were determined by liquid chromatography-mass spectrometry. Mean (+/-SE) concentrations of HMX in eggs were 21.0+/-5.9, 1113+/-79.0, 3864+/-154.0, and 7426+/-301.1 ng g(-1) in control, low, medium and high dose groups, respectively. There were significant differences in oxygen consumption among the three embryo ages, however differences among the ages were not consistent among dose groups (age x dose group interaction p<0.0001). Oxygen consumption rates did not vary as a function of HMX in embryos (p=0.18). No evidence was observed for alterations of in ovo metabolic rates associated with HMX exposure.

Cytisine for smoking cessation: a research agenda.

Cytisine has a molecular structure somewhat similar to that of nicotine and varenicline. The concept for the new smoking cessation drug varenicline was based partly on cytisine. Like varenicline, cytisine is a partial agonist of nicotinic acetylcholine receptors, with high affinity for alpha4beta2 receptors. Cytisine has been used since the 1960s as a smoking cessation drug in Eastern and Central Europe, but has remained largely unnoticed elsewhere. Three placebo-controlled trials, conducted in East and West Germany in the 1960s and 1970s, suggest that cytisine, even with minimal behavioural support, may be effective in aiding smoking cessation. Cytisine tablets are very inexpensive to produce and could be a more affordable treatment than nicotine replacement, bupropion and varenicline. There is however a dearth of scientific research on the properties of cytisine, including safety, abuse liability and efficacy. This paper seeks to identify research priorities for molecular, animal and clinical studies. In particular, new studies are necessary to define the nicotinic receptor interaction profile of cytisine, to establish its pharmacokinetics and pharmacodynamics in humans, to determine whether animals self-administer cytisine, and to ascertain whether cytisine is safe and effective as a smoking cessation drug. Potentially, this research effort, contributing to wider use of an inexpensive drug, could save many lives.

Effects of HMX-lead mixtures on reproduction of the earthworm Eisenia andrei.

High metal (e.g., Pb) concentrations are typically found in explosive-contaminated soil, and their presence may increase, decrease, or not influence toxicity predicted on the basis of one explosive alone (e.g., HMX). Nevertheless, few data are available in the scientific literature for this type of multiple exposure. Soil organisms, such as earthworms, are one of the first receptors affected by the contamination of soil. Therefore, a reproductive study was conducted using Eisenia andrei in a forest-type soil. Both HMX and Pb decreased reproduction parameters (number of total cocoons, hatched cocoons, and surviving juveniles) individually. Based on the total number of cocoons, HMX was more toxic in a forest soil than Pb, with EC(50) of 31 mg kg(-1), and 1068 mg kg(-1), respectively. The slope of the concentration-response curve was significantly greater in the case of Pb, which is consistent with the possibility that the two compounds do not act on the same target site. The response-addition model was used to predict the response of earthworms and to test for interaction between the two contaminants. The predicted toxicity was not significantly different than the observed toxicity, implying that Pb and HMX were considered noninteractive compounds. The combined action of Pb-HMX may be described, therefore, as dissimilar-noninteractive joint action in a forest soil. The results illustrate the relevance of considering the presence of metals in the risk assessment of explosive-contaminated sites because metals can add their toxicity to explosives. Extension of this study to other types of soil and other metals would improve the understanding of toxicity at these sites.

Explosives: fate, dynamics, and ecological impact in terrestrial and marine environments.

An explosive or energetic compound is a chemical material that, under the influence of thermal or chemical shock, decomposes rapidly with the evolution of large amounts of heat and gas. Numerous compounds and compositions may be classified as energetic compounds; however, secondary explosives, such as TNT, RDX, and HMX pose the largest potential concern to the environment because they are produced and used in defense in the greatest quantities. The environmental fate and potential hazard of energetic compounds in the environment is affected by a number of physical, chemical, and biological processes. Energetic compounds may undergo transformation through biotic or abiotic degradation. Numerous organisms have been isolated with the ability to degrade/transform energetic compounds as a sole carbon source, sole nitrogen source, or through cometabolic processes under aerobic or anaerobic conditions. Abiotic processes that lead to the transformation of energetic compounds include photolysis, hydrolysis, and reduction. The products of these reactions may be further transformed by microorganisms or may bind to soil/sediment surfaces through covalent binding or polymerization and oligomerization reactions. Although considerable research has been performed on the fate and dynamics of energetic compounds in the environment, data are still gathering on the impact of TNT, RDX, and HMX on ecological receptors. There is an urgent need to address this issue and to direct future research on expanding our knowledge on the ecological impact of energetic transformation products. In addition, it is important that energetic research considers the concept of bioavailability, including factors influencing soil/sediment aging, desorption of energetic compounds from varying soil and sediment types, methods for modeling/predicting energetic bioavailability, development of biomarkers of energetic exposure or effect, and the impact of bioavailability on ecological risk assessment.

Bioaccumulation of explosive compounds in the marine mussel, Mytilus galloprovincialis.

The bioaccumulative potential of the explosive compounds, 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) were assessed in water only exposures with the Mediterranean mussel (Mytilus galloprovincialis). Toxicokinetics experiments provided uptake rates, elimination rates, biological half-lives, and bioconcentration factors (BCFs). Kinetic BCFs were 1.61, 0.87, and 0.44, for TNT, RDX, and HMX, respectively, and confirmed the expected low bioaccumulative potential of these weakly hydrophobic compounds based on logK(ow). Because apparent steady-state conditions were observed within the 4h uptake period, steady-state BCFs were also calculated, and were within 20% of kinetic BCFs. TNT was rapidly biotransformed to aminodinitrotoluenes within minutes, while no transformation products were measured for RDX or HMX. Uptake clearance rates varied among the compounds, while elimination rates and associated half-lives were extremely fast (0.15-0.49h). It is unlikely, based on these data, that exposure conditions for these explosive compounds in the marine environment pose unacceptable risks to mussels, and it appears that potential for trophic transfer is quite low.

Toxicity of explosive compounds to the marine mussel, Mytilus galloprovincialis, in aqueous exposures.

Lethal and sublethal effects of the explosive compounds, 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) were assessed in separate water only exposures to the Mediterranean mussel (Mytilus galloprovincialis). Toxicity endpoints included survival and byssal thread formation in adults, and larval development success of embryos, in 96- and 48-h exposures, respectively. The larval development endpoint was over an order of magnitude more sensitive to TNT compared to adult survival, with median effective concentration (EC50) values of 0.75 and 19.5mgL(-1) (3.30 and 74.1micromolL(-1)), respectively. Byssal thread formation (48h EC50=6.57mgL(-1); 29.3micromolL(-1)) was also impaired at sublethal concentrations. The highest RDX and HMX concentrations tested (28.4 and 1.9mgL(-1) [124 and 6.43micromolL(-1)], respectively) failed to promote any significant toxicological effect in exposed mussels. Median lethal residues (LR50) of 14.0microg g(-1) (51.0nmolg(-1)) ww for TNT in the adults were similar to those measured for other marine invertebrates in previous studies, while body residues as high as 19.6 and 0.92microg g(-1) (86 and 3.1nmolg(-1)) ww were not associated with any toxicity for RDX and HMX, respectively.

Effects of octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) exposure on reproduction and hatchling development in Northern bobwhite quail.

Adult Northern bobwhite quail (Colinus virginianus) were exposed via food to octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX), an energetic compound found in soils at military training installations. Depuration of HMX into eggs was examined in an initial study, and effects on egg production, hatching, growth, development, and survival of chicks were examined in a follow-up study. HMX was readily and rapidly transferred from female quail into eggs. Marked weight loss was observed in quail exposed to 125 and 250 mg/kg HMX in food, likely due to reductions in food intake rather than a toxic mechanism. In the second study, significant alterations in body mass occurred among quail at concentrations >52.5 +/- 9.3 mg/kg but not at 12.3 +/- 1.1 mg/kg in food. Treatment-related reductions in food consumption and decreases in egg laying rates were observed. No HMX-related effects were found in chick growth or survival. Quail inhabiting HMX-contaminated sites could possibly be exposed to HMX and therefore deposition of HMX into eggs is also possible. However, results of these studies further suggest that the potential for reproductive toxicity of HMX to birds is low.