Evaluation of microRNAs-208 and 133a/b as differential biomarkers of acute cardiac and skeletal muscle toxicity in rats.

Conventional circulating biomarkers of cardiac and skeletal muscle (SKM) toxicity lack specificity and/or have a short half-life. MicroRNAs (miRNAs) are currently being assessed as biomarkers of tissue injury based on their long half-life in blood and selective expression in certain tissues. To assess the utility of miRNAs as biomarkers of cardiac and SKM injury, male Sprague-Dawley rats received a single dose of isoproterenol (ISO); metaproterenol (MET); allylamine (AAM); mitoxantrone (MIT); acetaminophen (APAP) or vehicle. Blood and tissues were collected from rats in each group at 4, 24 and 48h. ISO, MET, and AAM induced cardiac and SKM lesions and APAP induced liver specific lesions. There was no evidence of tissue injury with MIT by histopathology. Serum levels of candidate miRNAs were compared to conventional serum biomarkers of SKM/cardiac toxicity. Increases in heart specific miR-208 only occurred in rats with cardiac lesions alone and were increased for a longer duration than cardiac troponin and FABP3 (cardiac biomarkers). ISO, MET and AAM induced increases in MyL3 and skeletal muscle troponin (sTnl) (SKM biomarkers). MIT induced large increases in sTnl indicative of SKM toxicity, but sTnl levels were also increased in APAP-treated rats that lacked SKM toxicity. Serum levels of miR-133a/b (enriched in cardiac and SKM) increased following ISO, MET, AAM and MIT treatments but were absent in APAP-treated rats. Our results suggest that miR-133a/b are sensitive and specific markers of SKM and cardiac toxicity and that miR-208 used in combination with miR-133a/b can be used to differentiate cardiac from SKM toxicity.

Paving the Route to Plasma miR-208a-3p as an Acute Cardiac Injury Biomarker: Preclinical Rat Data Supports Its Use in Drug Safety Assessment.

Drug-induced cardiac injury (DICI) detection remains a major safety issue in drug development. While circulating microRNAs (miRs) have emerged as promising translational biomarkers, novel early detection biomarkers of cardiotoxicity are needed. This work aims at evaluating whether a panel of putative cardiac injury plasma miRs could serve as early DICI biomarkers in a 4-day rat preclinical model. Out of a panel of 68 selected targets, we identified plasma miR-208a-3p as being significantly upregulated after single administration with either isoproterenol (ISO) or allylamine (AAM). This provides the first evidence of miR-208a-3p detection after AAM administration. Moreover, similarly to cardiac troponins (cTn), plasma miR-208a-3p expression profile appears to be compound-specific with most significant early changes occurring in ISO-treated rats. Overall, miR-208a-3p performance in detecting the severity of myocardial injury, as well as the magnitude of miR-208a-3p increase after ISO or AAM administration, were comparable to that of cTn. Our results highlight the importance of assessing the whole time-dependent profiles of miR expression. Hence, time course evaluation revealed plasma miR candidates whose expression was not stable across the duration of the study in the vehicle group, restricting their utility as cardiac injury-specific biomarkers. In light of these findings, miR-208a-3p has a potential to complement the existing biomarkers of cardiac injury specifically in the context of evaluating toxicity in a time-dependant manner. Assessment of miR-208a-3p in other DICI settings would strengthen its robustness as an early detection biomarker leading to a warranted extensive and rigorous validation.

Binding mode of CpG oligodeoxynucleotides to nanoparticles regulates bifurcated cytokine induction via Toll-like receptor 9.

The interaction of cytosine-phosphate-guanine oligodeoxynucleotides (CpG ODNs) with Toll-like receptor 9 (TLR9) activates the immune system. Multimeric class A CpG ODNs induce interferon-α (IFN-α) and, to a lesser extent, interleukin-6. By contrast, monomeric class B CpG ODNs induce interleukin-6 but not IFN-α. This difference suggests that the multimerization of CpG ODN molecules is a key factor in IFN-α induction. We multimerized class B CpG ODN2006x3-PD molecules that consist entirely of a phosphodiester backbone onto quantum dot silicon nanoparticles with various binding modes. Herein, we present the binding mode-dependent bifurcation of cytokine induction and discuss its possible mechanism of CpG ODN and TLR9 interaction. Our discoveries also suggest that nanoparticles play roles in not only delivery of CpG ODNs but also control of CpG ODN activity.

Naftifine-analogues as anti-Trypanosoma cruzi agents.

Chagas disease represents a relevant health problem in Central and South America. The first line of treatment is Nifurtimox and Benznidazole which have a great deal of disadvantages that demands the rapid generation of therapeutic alternatives. Based in our research on aza-thiaheterocycles as anti-Trypanosoma cruzi agents we identified pharmacophores that act through oxidative stress. Here, we describe the synthesis and the activity of new containing bioactive-heterocycles analogues of naftifine as potential T. cruzi membrane sterol biosynthesis inhibitors. Benzimidazole 1,3-dioxides (11 and 13) and quinoxaline 1,4-dioxides (22 and 23) displayed excellent parasite/mammal selectivity indexes. Analysis of the free sterols from parasite incubated with the compounds showed that any of them are able to accumulate squalene suggesting that in the anti-T. cruzi mechanism of action is not involved the inhibition of sterol biosynthesis. Some derivatives were also tested as antifungal agents. The results obtained in the present work open potential therapeutic possibilities of new compounds for these infectious diseases.

In vivo investigation of the inflammatory response against allylamine plasma polymer coated titanium implants in a rat model.

Titanium (Ti) is an established biomaterial for bone replacement. However, facilitation of osteoblast attachment by surface modification with chemical groups could improve the implant performance. Therefore, this study aimed to evaluate the effect of a plasma polymerized allylamine (PPAAm) layer on the local inflammation in a rat model. Three series (RM76AB, RM78AB, RM77AB) of PPAAm-treated Ti plates were prepared using different plasma conditions. Twelve male LEW.1A rats received one plate of each series and one uncoated control plate implanted into the back musculature. After 7, 14 and 56 days, four rats were euthanized to remove the implants with surrounding tissue. Total monocytes/macrophages, tissue macrophages, T-cells and MHC-class-II-positive cells were morphometrically counted. On day 14, the macrophage/monocyte number was significantly higher for the controls than for the PPAAm samples. On day 56, the RM76AB and RM78AB samples had significantly lower numbers than RM77AB and the controls. The same was found for the tissue macrophages. No change over time and no differences between the implants were found for the T-cells. For the number of MHC-class-II-positive cells, a significant decrease was found only for the RM78AB implants between day 14 and day 56. Physico-chemical analysis of the PPAAm implants revealed that the RM77AB implants had the lowest water absorption, the highest nitrogen loss and the lowest oxygen uptake after sonication. These results demonstrate that the PPAAm samples and the controls were comparable regarding local inflammation, and that different plasma conditions lead to variations in the material properties which influence the tissue reaction.

A fluorescence-based screening assay for DNA damage induced by genotoxic industrial chemicals.

A rapid screening assay to detect chemically-induced DNA damage resulting from exposure of surrogate DNA to genotoxic compounds is reported. This assay is based on changes in the melting and annealing behavior observed for damaged DNA. Exposure of calf thymus DNA to genotoxic industrial chemicals reduced the extent to which the DNA annealed as measured using a double strand DNA selective fluorescent indicator dye. Formaldehyde, acrolein, crotonaldehyde and bromoethane showed the most prominent effects, chloroacetone and allylamine exhibited lesser effects, and acryrlonitrile showed no statistically significant assay response. The assay response for formaldehyde and crotonaldehyde were measured over the concentration range of 10-100 mM and 50-300 mM, respectively. This assay showed little response for the cytotoxic compounds phenol, cyclohexane and toluene but was sensitive to the effects of DNA damaging compounds such as mitomycin C and glutaraldehyde.

Colesevelam hydrochloride does not cause maternal or fetal toxicity in rats and rabbits.

WelChol (colesevelam hydrochloride), a bile acid sequestrant for the treatment of hypercholesterolemia, was evaluated for adverse effects on reproduction and fetal development using standard preclinical tests. During gestation, Sprague-Dawley rats used in the developmental toxicity study received feed, feed/control article or feed plus 300, 1,000 or 3,000 mg/kg/day colesevelam whereas rats in the pre- and postnatal toxicity study received vehicle or 100, 300 or 1,000 mg/kg/day colesevelam via gavage. New Zealand white rabbits received control or 100, 500 or 1,000 mg/kg/day colesevelam via gavage. No deaths, premature deliveries or gross pathologic lesions were observed up to gestation day (GD) 20 for rats and GD 28 for rabbits. No significant differences in the number of pregnant animals, average litter size, percentage of viable fetuses, fetal body weights, number of corpora lutea, fetal viability, or gross malformations were observed versus controls. Pre- and postnatal effects were assessed in pregnant rats receiving 100, 300 or 1,000 mg/kg/day colesevelam from GD 6 to postpartum day 22. Gestation, parturition and lactation in F(0) generation dams were similar between treatment and control groups. Colesevelam did not affect physical or neurological development or induce gross pathological changes in F(1) generation rats. Colesevelam does not produce developmental toxicity in rats or rabbits, nor does it exhibit pre- or postnatal toxicity in rats at the tested doses.

Modulation of alpha4 integrin mRNA levels is coupled to deficits in vasomotor function in rat arterioles by allylamine.

Allylamine, a selective cardiovascular toxin that induces oxidative stress, is known to alter expression of extracellular matrix and cell adhesion proteins that are central to arterial remodeling. Our goals were to determine whether AAM treatment in rats modulates integrin/matrix-dependent arteriolar function, and to what extent integrin expression correlated to these alterations. Integrins are transmembrane proteins that facilitate mechanical and molecular signaling between the extracellular matrix and cytoskeleton, and so are suitable candidates for involvement in phenotypic and functional alterations of smooth muscle in response to oxidative stress. Arg-Gly-Asp (RGD) and Leu-Asp-Val (LDV), two integrin-binding motifs found in ECM proteins such as collagens and fibronectin, are known to interact with integrins alphavbeta3 and alpha4beta1, respectively. Previously, we found that RGD containing peptides induce vasodilation through alphavbeta3, while LDV containing peptides induce vasoconstriction through alpha4beta1 of normal rat cremasteric arterioles. In allylamine-treated rats (AAM), the vasomotor response to LDV, but not RGD, was attenuated in a dose-dependent manner. To determine whether changes in integrin subunit mRNA levels correlated with these functional changes, we performed reverse transcription and Real-time PCR for alpha4 and beta3 integrin subunits on RNA isolated from single, first-order cremasteric arterioles. AAM treatment caused a dose-dependent decrease in alpha4 mRNA expression, but not beta3 mRNA expression, suggesting that the changes in vasomotor activity to LDV peptides may be attributable in part to reduced alpha4 expression upon exposure to AAM. These data are supported by similar decreases in alpha4integrin cell surface protein expression in cultured vascular smooth muscle cells treated either in vivo and in vitro with AAM.

Modulation of cyclin dependent kinase inhibitor proteins and ERK1/2 activity in allylamine-injured vascular smooth muscle cells.

Chronic oxidative injury by allylamine (AAM) induces proliferative vascular smooth muscle cell (vSMC) phenotypes in the rat aorta similar to those seen in rodent and human atherosclerotic lesions. The proliferative advantage of AAM vSMC compared to control cells is maintained with serial passage of the cells and the advantage is nullified when AAM cells are seeded on a collagen substrate. In this study, we evaluate the potential role of cyclin dependent kinase inhibitors, p27 and p21, and mitogen activated protein (MAP) kinases, ERK1/2, in mediating the proliferative advantage of AAM stressed vSMC over control cells on plastic or collagen substrates. p27 levels in randomly cycling cells were comparable in both cell types irrespective of the substrate. In contrast, basal levels of p21 were 1.9 +/- 0.3 (P < 0.05)-fold higher in randomly cycling AAM cells seeded on plastic compared to controls, a difference that was lost on a collagen substrate. Following G0 synchronization, basal levels of both p27 and p21 were higher in AAM cells seeded on plastic compared to controls (1.7 +/- 0.2 and 2.0 +/- 0.3-fold, respectively, P < 0.05), but these differences were lost upon mitogenic stimulation. Pyrrolidine dithiocarbamate (PDTC) decreased p27 and p21 levels in cycling AAM cells relative to controls in a substrate-dependent manner. AAM cells seeded on plastic exhibited enhanced ERK1/2 activation upon mitogenic stimulation; seeding on collagen nullified this advantage. The duration of ERK1/2 activation was prolonged in AAM cells independently of the seeding substrate. We conclude that substrate-dependent acquisition of proliferative phenotypes following repeated cycles of AAM injury correlates with modulation of the cyclin dependent kinase inhibitors, p27 and p21.

Dietary administration of colesevelam hydrochloride does not affect fertility or reproductive performance in rats.

Colesevelam hydrochloride (HCl) (WelChol; Sankyo Pharma) is a novel, highly potent, bile acid-binding polymer used for the treatment of hypercholesterolemia. The primary aim of this study was to determine the effects of dietarily administered colesevelam HCl on fertility and reproductive performance parameters. To assess these effects, sexually mature Sprague-Dawley rats were randomized to one of five treatment groups: feed alone, feed plus control article (SigmaCell), or feed plus colesevelam HCl 200, 1000, or 2000 mg/kg/day. Male and female rats were administered the appropriate group agent for 28 and 15 days, respectively, and were subsequently paired together for cohabitation and mating. Females continued to receive the test agent in their dietary formulation through presumed gestation day (GD) 7. Presumed pregnant females underwent cesarean section on GD 20. Food consumption rate, body weight, gross necropsy, and standard preclinical tests for reproduction and fertility were performed for each test animal. No statistically significant differences were found between control and drug-treated groups for any tested endpoints of reproduction. All animals placed in cohabitation successfully mated. Uterine and litter end points were unaffected by dosages of colesevelam HCl as high as 2000 mg/kg/day. There were no significant differences between treatment group litter averages in the number of corpora lutea, implantation sites, litter size, live fetuses, body weights, early/late resorptions, and the number of dams with viable fetuses. In addition, no external alterations of fetal morphology were attributable to treatment with colesevelam HCl when administered up to the embryo implantation stage. In male animals, no significant differences were found between the colesevelam HCl and control study groups in the average caudal epididymal sperm count or sperm concentration, total number of motile and nonmotile sperm, and the total percentage of motile sperm. Based on these data, colesevelam HCl does not have any significant adverse reproductive or fertility effects in rats, even when administered at doses approximately 30 times greater than the approved clinical dose.

Amine metabolism: a novel path to coronary artery vasospasm.

We hypothesized that allylamine (AA) induces subendocardial necrosis in mammals via coronary artery (CA) vasospasm. Additionally, AA toxicity is likely dependent on the enzyme semicarbazide-sensitive amine oxidase (SSAO), which is highly expressed in the aorta of rats and humans. We tested whether AA or acrolein (1, 10, 100, and 1000 microM), a highly reactive product of AA metabolism by SSAO, could contract CA or thoracic aorta (TA) in vitro and if the AA effects involved SSAO. AA or acrolein produced a similar pattern of responses in both CA and TA rings at 100 and 1000 microM, including (1) increased basal tension, (2) enhanced agonist-induced contraction (hypercontractility or vasospasm), (3) remarkable, agonist-induced slow wave vasomotion (vasospasm), and (4) irreversible reduction in vessel contractility after 1 mM exposure. Endothelium-dependent acetylcholine-induced relaxation was not altered during vasospasm in either vessel. Pretreatment with the SSAO inhibitor semicarbazide (1 mM; 10 min) prevented or significantly reduced the majority of AA's effects in both CA and TA rings and inhibited 100% of the SSAO activity present in rat TA and human CA and TA. We propose a two-step model for AA induction of CA vasospasm and resultant myocardial necrosis: (1) metabolism of AA to acrolein by coronary arterial SSAO activity and (2) acrolein induction of CA vasospasm independent of endothelial injury-a novel path.

The role of plasma semicarbazide-sensitive amine oxidase in allylamine and beta-aminopropionitrile cardiovascular toxicity: mechanisms of myocardial protection and aortic medial injury in rats.

Allylamine (AA; 3-aminopropene) and beta-aminopropionitrile (betaAPN) combined treatment (AA + betaAPN) results in myocardial protection from AA-induced subendocardial necrosis and a rapid and extensive aortic medial smooth muscle injury in rats. To determine the mechanisms of AA + betaAPN-induced vascular toxicity, cardiovascular parameters were monitored during a 10-day exposure by gavage in male Sprague-Dawley rats (180-200 g). Water intake and urine output were measured in rats treated with water, AA (100 mg kg(-1) body weight), betaAPN (1 g kg(-1) body weight), and AA + betaAPN for 10 days in metabolic cages. Plasma and urine samples were analyzed for blood urea nitrogen, CO2, creatinine, hematocrit, electrolytes (Na+, K+, Cl-), and osmolality. Heart and plasma semicarbazide-sensitive amine oxidase metabolic capacity (SSAO)was also measured following 1, 3 and 10 days of treatment. Following 10 day exposure to control or AA + betaAPN treatment, thoracic aortic rings (approximately 3 mm) were removed, and aortic reactivity to contractile and relaxant agonists was tested in vitro. In addition, cultured rat aorta vascular smooth muscle cells or rat heart beating myocytes were exposed to various concentrations of AA and betaAPN or AA metabolites and betaAPN to test for synergism in vitro. Several of the changes in in vivo cardiovascular parameters were shared, both in direction and magnitude, between the AA + betaAPN and the AA alone or the betaAPN alone treatments. This suggests that these effects (e.g. increased water intake and urine flow, decreased hematocrit, decreased heart and plasma SSAO metabolic capacity) were dependent on an AA alone or a betaAPN alone effect and were not AA + betaAPN specific effects. Significant inhibition of plasma and heart SSAO metabolic capacity occurred in the betaAPN alone and the AA + betaAPN treatments, but not in the AA alone treatment. Aortic rings from AA + betaAPN treated rats were contracted significantly less than anatomically-matched control rat aortic rings by 100 mM potassium chloride or by 10 microM norepinephrine. BetaAPN offered substantial protection against AA cytotoxicity in cultured vascular smooth muscle cells and beating myocytes, but did not alter the cytotoxicity of AA metabolites (i.e. acrolein, H2O2, or ammonia) in vascular smooth muscle cells as determined by the MTT viability assay. Overall, these data suggest that myocardial protection from AA injury that occurs in the combined AA + betaAPN treatment is likely due to inhibition of plasma SSAO. This may result in an increase in the AA dose accumulation and metabolism in the aorta leading to the severe aortic medial injury.

Developmental vasculotoxicity associated with inhibition of semicarbazide-sensitive amine oxidase.

The endogenous substrate(s) and physiological function(s) of semicarbazide-sensitive amine oxidase (SSAO), a group of enzymes exhibiting highest activity in vascular smooth muscle cells of the mammalian aortic wall, remain undetermined. This study examines the pathophysiological effects in the thoracic aortic wall resulting from specific in vivo SSAO inhibition. Weanling Sprague-Dawley rats were treated acutely or chronically with either semicarbazide hydrochloride or the allylamine derivatives MDL-72274 or MDL-72145 (Marion Merrell Dow Research Institute, Cincinnati, OH). Treatment with these compounds produced acute (6 and 24 h) and chronic (21 day) lowering of SSAO activity in aorta and lung with little effect on the activity of the vital matrix-forming enzyme, lysyl oxidase, in aortas of chronically treated animals. Chronic SSAO inhibition produced lesions consisting of striking disorganization of elastin architecture within the aortic media accompanied by degenerative medial changes and metaplastic changes in vascular smooth muscle cells. No significant difference in the total weight of dry, lipid-extracted aortic elastin and collagen components were observed between chronically SSAO inhibited and control animals. However, the amount of mature elastin was lowered and mature collagen was raised in the aortas of animals treated chronically with semicarbazide. Descending thoracic aortic rings isolated from chronically SSAO-inhibited animals had larger cross-sectional diameters (i.e., exhibited dilation) when compared to corresponding rings from control animals. This study demonstrates that developmental toxicity, characterized by striking vascular lesions and dilated thoracic aortas, can result from specific in vivo SSAO inhibition, suggesting a role for SSAO in connective tissue matrix development and maintenance, and specifically in the development of normal elastin.

Cytotoxic and anticancer activities of some 1-aryl-2-dimethylaminomethyl-2-propen-1-one hydrochlorides.

A series of 1-aryl-2-dimethylaminomethyl-2-propen-1-one hydrochlorides demonstrated marked cytotoxicity towards approximately 55 human tumour cell lines from different neoplastic diseases. In general they were more potent than melphalan and displayed selective toxicity towards human leukemic cells. A representative compound, 1-phenyl-2-dimethyl-aminomethyl-2-propen-1-one hydrochloride (2a), had similar cytotoxicity as melphalan towards murine P388 and L1210 leukemic cells. In addition, 2a reduced the sizes of a number of human tumour xenografts including colon, prostatic and melanotic cancers passaged in athymic mice. Compound 2a showed excellent activity towards Ehrlich ascites carcinoma and B16F1 melanoma in mice which was enhanced using niosomes. One may conclude from the data generated that 1-aryl-2-dimethylaminomethyl-2-propen-1-one hydrochlorides are a novel series of cytotoxic and anticancer agents.

The role of glutathione S-transferases as a defense against reactive electrophiles in the blood vessel wall.

The glutathione transferases (GSTs) are a family of ubiquitous enzymes that catalyze the conjugation of reduced glutathione (GSH) with reactive electrophiles. Rat vascular tissue contains GST isoforms that represent a major cellular defense mechanism against atherogenic alpha,beta-unsaturated aldehydes (Misra et al., Toxicol. Appl. Pharmacol. 133, 27-33, 1995). In this study we examined the role of GSTs in providing protection to cultured neonatal vascular smooth muscle cells (VSMCs) from the alpha,beta-unsaturated carbonyl cardiovascular toxins, allylamine and its metabolite, acrolein. Confluent cultured cells were exposed to 2 to 10 microM allylamine (a cardiovascular toxin that is metabolized in vivo and in vitro by VSMCs to the reactive aldehyde, acrolein) or to acrolein (2-10 microM) for 48 h; dose-cytotoxicity curves were generated utilizing a tetrazolium-dependent cytotoxicity assay. Concommittant treatment with sulfasalazine, an established inhibitor of GST, was found to markedly increase allylamine- or acrolein-induced cytotoxicity, decreasing the LC50 by two- to threefold at 50 to 100 microM sulfasalazine. A clonogenic survival assay in VSMCs exposed to these compounds for 4 h confirmed lethal toxicity and enhanced toxicity following cotreatment with sulfasalazine. Isobologram analysis (which statistically defines the limits of additivity of two independent treatments) showed that the sulfasalazine effect on both allylamine and acrolein cytotoxicity was supraadditive, or synergistic. Sulfasalazine was not cytotoxic to VSMCs in the range of concentrations that augmented acrolein or allylamine cytoxicity; total GST activity was inhibited, however, in a dose-dependent manner in that range. GST purified by GSH-affinity chromatography from pelleted untreated cells gave specific activities and kinetic constants consistent with those previously reported for rat aorta total GSTs. The catalytic efficiency (Kcat/Vm) was found to be much greater for 4-hydroxy-2-nonenal than for 1-chloro-2,4-dinitrobenzene (0.058 vs 0.4 s-1 mM-1). Western blot of purified total GSTs using antibodies against rec-mGSTA4-4 revealed a single band at 25 kDa, confirming the presence of a GST isozyme immunologically similar to rat GST8-8, which is known to utilize alpha,beta-unsaturated carbonyls as preferred substrates. Our data indicate that GSTs are an important defense in the vascular media, protecting blood vessels against alpha,beta-unsaturated carbonyl cardiovascular toxins that are involved in initiating atherosclerotic lesions.

Activity of N,N-dimethy-1-2-propen-1-amine derivatives in mice experimentally infected with Trypanosoma cruzi.

Propenamine derivatives 3-(4'-bromo-[1,1'-biphenyl]-4-yl)-3-(4-X-phenyl)-N,N-dimethyl-2-prope n-1-amine (where X = H or Br) were tested in experimentally infected mice with Trypanosoma cruzi (Y strain). When a daily dose of 20 mg kg-1 of the bromo (X = Br) derivative for 9 consecutive days was used, no parasite by optical microscopy was found. Significant parasitemic decrease was also observed with a single dose (100 mg kg-1) of this compound. Moreover, both treatment schemes displayed a strong protective effect characterized by decreased of mortality. On the other hand, similar treatment schedules using the unsubstituted (X = H) derivative led to parasitemic and mortality curves similar to the control group. These results indicate that the bromo derivative has a remarkable activity against T. cruzi infection. Due to the potentiality of this derivative, further investigation of this class of compounds as chemotherapeutic agents for Chagas' disease is of prime importance.

Allylamine and beta-aminopropionitrile induced aortic medial necrosis: mechanisms of synergism.

We have developed a model of aortic smooth muscle necrosis in adult Sprague Dawley rats by feeding them two vascular toxins (allylamine HCl, or AA, and beta-aminopropionitrile, or betaAPN) in concert for 10 days. Either toxin given alone does not cause aortic lesions. In order to shed light on the mechanism of the synergistic action of these two toxins we fed known modulators of AA or betaAPN toxicity to rats concurrently with the two toxins. As modulators we used (a) semicarbazide (98 mg/kg/day, given 4 h prior to toxins), a known inhibitor of the vascular enzyme SSAO which metabolizes AA; (b) L-cysteine (1.5% in rat chow, beginning 3 days prior to toxins), which has been shown to reduce the toxic effects of betaAPN; and (c) phenelzine sulphate (3 mg/kg/day, given 4 h prior to toxins), an inhibitor of SSAO and potentiator of betaAPN toxicity. Rats were fed various combinations of the toxins and modulators by gavage: water (n = 8); (AA, 100 mg/kg/day) AA + phenelzine (n = 8); AA + semicarbazide (n = 8); AA + L-cysteine (n = 11); (betaAPN, 1 g/kg/day) betaAPN + phenelzine (n = 8); betaAPN + semicarbazide (n = 8); betaAPN + L-cysteine (n = 8); (AA, 100 mg + betaAPN, 1 g/kg/day) AA + betaAPN + phenelzine (n = 9), AA + betaAPN + semicarbazide (n = 8); AA + betaAPN + L-cysteine (n = 12); phenelzine (3 mg/kg/day) (n = 4); semicarbazide (98 mg/kg/day) (n = 4) and L-cysteine (1.5% in rat chow) (n = 4). We found that phenelzine sulphate (a drug previously used in the treatment of hypertension) when given with AA reproduced the AA + betaAPN induced aortic lesions. Phenelzine + betaAPN caused no lesions, but when combined with AA + betaAPN, aortic lesions were intensified and included marked secondary degeneration of the vascular wall. Semicarbazide was found to completely obviate the vascular toxicity of AA + betaAPN. L-Cysteine feeding markedly decreased the incidence and severity of vascular lesions in AA + betaAPN treated rats, but did not change the incidence or severity of heart lesions caused by AA alone. These data indicate that the synergistic necrotizing toxicity of AA + betaAPN is primarily an AA effect. We postulate that some modulating influence of betaAPN (or phenelzine) on tissue distribution, metabolism, or detoxification pathways of AA increases AA's acute vascular toxicity, whereas semicarbazide offers protection by inhibiting the initial deamination of AA to a highly reactive aldehyde.

Allylamine cardiovascular toxicity: evidence for aberrant vasoreactivity in rats.

Allylamine (AA, 3-aminopropene) is a specific cardiovascular toxin used experimentally to model myocardial necrosis and atherosclerosis. In these physiologic experiments, 10-day AA exposure (100 mg . kg-1 . day-1 by gavage) produced severe myocardial necrosis and increased heart rate but did not affect systolic blood pressure in rats. Mid-thoracic aortic ring segments were removed, and reactivity to contractile and relaxant agonists was tested. Aortic rings (approximately 3 mm) from AA-treated rats were contracted significantly more by high potassium (100 mM) and slightly more by norepinephrine (NE, 10 microM) than anatomically matched control aortic rings. No difference in aortic ring NE sensitivity or percentage relaxation in response to acetylcholine (1 microM) or sodium nitroprusside (100 microM) was detected between control and AA-treated rat aortic rings. Allylamine (1 microM-1 mM) induced modest, concentration-dependent contractions and tension oscillations in aortic rings from both control and AA-treated rats. Aortic rings from AA-treated rats, however, were more sensitive to AA. Vascular smooth muscle cells derived from control and AA-treated rat aortas had similar toxic sensitivity to AA in vitro using the MTT viability assay. The mechanisms by which AA exposure increased heart rate in vivo and contractility of aortic rings are unknown. These experiments support the previously proposed concept that AA-induced acute myocardial necrosis is due to coronary vasospasm and myocardial ischemia and cell injury.

Allylamine and beta-aminopropionitrile-induced vascular injury: enhanced expression of high-molecular-weight proteins.

In the present study we describe changes in aorta at the protein level associated with allylamine (AA) and beta-aminopropionitrile (beta APN) induced vascular toxicity in a rat model. This model represents a remarkable synergistic, necrotizing toxic effect of these combined toxins, and our rationale was to examine protein expression in order to shed light on the mechanisms underlying this synergism. Rats were given AA (100 mg/kg body weight/day) and beta APN (1 g/kg body weight/day) by gavage for 10 d; this protocol has been shown to result in smooth-muscle necrosis, but no visible connective tissue changes. Soluble and insoluble fractions from AA + beta APN- or from beta APN-treated aorta showed enhanced expression of three high-molecular-weight protein bands (ranges between approximately 120 and 95 kD). The time course of induction of proteins showed the appearance of AA + beta APN-induced specific proteins at d 3 of AA + beta APN treatment. Partial purification and characterization suggested that AA + beta APN specific proteins are likely to be collagen proteins (type I). Thus, the data presented in this article help in understanding the vascular toxicity induced by AA + beta APN or by beta APN, in that we have described an altered phenotypic expression of collagenous proteins indicative of selective medial vascular toxicity.

Contribution of serum and cellular semicarbazide-sensitive amine oxidase to amine metabolism and cardiovascular toxicity.

Semicarbazide-sensitive amine oxidase (SSAO) plays a role in the in vivo and in vitro toxicity of several environmental and endogenous amines. We investigated the role of SSAO as a component of cell culture medium (through addition of fetal calf serum (FCS)) compared to intracellular SSAO in the in vitro cytotoxicity of three amines and metabolites. Smooth muscle cells and beating cardiac myocytes were grown in 96-well plates and exposed to various concentrations and combinations of FCS in medium, amines (allylamine, AA; benzylamine, BZA; and methylamine, MA), and amine metabolites (aldehydes: acrolein, benzaldehyde, and formaldehyde; hydrogen peroxide, H2O2; ammonia, NH3). Amine and amine metabolite cytotoxicity was quantified by monitoring cell viability. SSAO activity was measured in FCS, cardiovascular cells, or rat plasma by a radioenzymatic assay using [14C]BZA. Our data show that AA and its aldehyde metabolite, acrolein, were the most toxic compounds to both cell types. However, AA toxicity was FCS-dependent in both cell types, while BZA, MA, and amine metabolite (i.e., aldehydes, H2O2, and NH3) cytotoxicity showed little FCS dependence. In these experiments, medium containing 10% FCS had a calculated amine metabolic capacity that was 30- to 50-fold that of the cultured smooth muscle cellular content in a single well of a 96-well plate. Our study demonstrates that SSAO in FCS contributes to amine metabolism and cytotoxicity to rat cardiovascular cells in vitro and how critical it is to evaluate serum for its role in mechanisms of amine toxicity in vitro and in vivo.