A zebrafish-based acoustic motor response (AMR) assay to evaluate chemical-induced developmental neurotoxicity.

Behavioral assays using early-developing zebrafish (Danio rerio) offer a valuable supplement to the in vitro battery adopted as new approach methodologies (NAMs) for assessing risk of chemical-induced developmental neurotoxicity. However, the behavioral assays primarily adopted rely on visual stimulation to elicit behavioral responses, known as visual motor response (VMR) assays. Ocular deficits resulting from chemical exposures can, therefore, confound the behavioral responses, independent of effects on the nervous system. This highlights the need for complementary assays employing alternative forms of sensory stimulation. In this study, we investigated the efficacy of acoustic stimuli as triggers of behavioral responses in larval zebrafish, determined the most appropriate data acquisition mode, and evaluated the suitability of an acoustic motor response (AMR) assay as means to assess alterations in brain activity and risk of chemical-induced developmental neurotoxicity. We quantified the motor responses of 120 h post-fertilization (hpf) larvae to acoustic stimuli with varying patterns and frequencies, and determined the optimal time intervals for data acquisition. Following this, we examined changes in acoustic and visual motor responses resulting from exposures to pharmacological agents known to impact brain activity (pentylenetetrazole (PTZ) and tricaine-s (MS-222)). Additionally, we examined the AMR and VMR of larvae following exposure to two environmental contaminants associated with developmental neurotoxicity: arsenic (As) and cadmium (Cd). Our findings indicate that exposure to a 100 Hz sound frequency in 100 ms pulses elicits the strongest behavioral response among the acoustic stimuli tested and data acquisition in 2 s time intervals is suitable for response assessment. Exposure to PTZ exaggerated and depressed both AMR and VMR in a concentration-dependent manner, while exposure to MS-222 only depressed them. Similarly, exposure to As and Cd induced respective hyper- and hypo-activation of both motor responses. This study highlights the efficiency of the proposed zebrafish-based AMR assay in demonstrating risk of chemical-induced developmental neurotoxicity and its suitability as a complement to the widely adopted VMR assay.

A prostate-specific membrane antigen (PSMA)-targeted prodrug with a favorable in vivo toxicity profile.

Prostate-specific membrane antigen (PSMA) is a promising target for the treatment of advanced prostate cancer (PC) and various solid tumors. Although PSMA-targeted radiopharmaceutical therapy (RPT) has enabled significant imaging and prostate-specific antigen (PSA) responses, accumulating clinical data are beginning to reveal certain limitations, including a subgroup of non-responders, relapse, radiation-induced toxicity, and the need for specialized facilities for its administration. To date non-radioactive attempts to leverage PSMA to treat PC with antibodies, nanomedicines or cell-based therapies have met with modest success. We developed a non-radioactive prodrug, SBPD-1, composed of a small-molecule PSMA-targeting moiety, a cancer-selective cleavable linker, and the microtubule inhibitor monomethyl auristatin E (MMAE). SBPD-1 demonstrated high binding affinity to PSMA (Ki = 8.84 nM) and selective cytotoxicity to PSMA-expressing PC cell lines (IC50 = 3.90 nM). SBPD-1 demonstrated a significant survival benefit in two murine models of human PC relative to controls. The highest dose tested did not induce toxicity in immunocompetent mice. The high specific targeting ability of SBPD-1 to PSMA-expressing tumors and its favorable toxicity profile warrant its further development.

Malformations and mortality in zebrafish early stages associated with elevated caspase activity after 24 h exposure to MS-222.

Tricaine methanesulfonate (MS-222) is a commonly used anaesthetic agent for immobilization of aquatic species. However, delayed development and malformations have been observed in 24 hpf (hours post-fertilization) zebrafish embryos after long-term immobilization. Still, no comprehensive study has been described regarding zebrafish exposure to MS-222 during the first hours of development, which are one of the most sensitive life stages to toxicants. Therefore, this research aimed to assess the toxicity of a 24 h exposure to MS-222 on zebrafish embryonic development. Based on the MS-222 LC50, early blastula stage embryos (~2 hpf) were exposed to 0, 12.5, 25 and 50 mg L-1 for 24 h and then allowed to develop up to 144 hpf. The chromatographic analysis showed that this anaesthetic agent bioaccumulates in 26 hpf zebrafish larvae in a concentration-dependent manner. In addition, increased mortalities and skeletal abnormalities were observed at 144 hpf, namely in the highest tested concentration. Yet, no craniofacial anomalies were observed either by alcian blue or calcein staining methods. Independently of the tested concentration, decreased speed and distance travelled were perceived in 144 hpf larvae. At the biochemical level, decreased in vivo reactive oxygen species (ROS) generation and apoptosis was observed. Additionally, catalase activity was increased at 26 hpf while results of mRNA expression showed a decreased gclc transcript content at the same time-point. Overall, data obtained highlight the toxicological risk of MS-222 and support ROS-mediated cell death signalling changes through the elevation of catalase activity as an adaptative or protective response.

Chemicals Weaken Shoal Preference in the Rare Minnow Gobiocypris rarus.

Fish behavioral responses are sensitive to chemicals in the water. We tested rare minnow tested for their shoal preference, and the shoal (school) factors including nutritional status, body size, and shoal (school) size that can make their preference most stable were measured. Then shoal preference was measured again while fish and shoal were subjected to a concentration gradient of chemicals (cadmium ion [Cd2+ ], tricaine methanesulfonate [MS222], and p-chloroaniline). The results showed that single rare minnow preferred shoals over blank control tanks. In addition, this preference was most stable when the shoal was well fed and contained 20 individuals 2 cm long. Although there was no significant response after exposure to p-chloroaniline, the time spent from entering the tank to start moving decreased greatly at concentrations of Cd2+ >3 mg/L and MS222 >11 mg/L. The time the test fish spent close to the shoal significantly decreased at Cd2+ >3 mg/L, MS222 >11 mg/L, and p-chloroaniline >10 mg/L, and the frequency of boundary line crossing increased at the same concentrations. The behavioral parameters changed by 20, 5, and 8 min once the lowest-observed-effect concentrations of Cd2+ , MS222, and p-chloroaniline, respectively, were added. Our study provides useful information on rare minnow shoal preference that may be used for a biological early warning system. Environ Toxicol Chem 2020;39:2018-2027. © 2020 SETAC.

MS-222 induces biochemical and transcriptional changes related to oxidative stress, cell proliferation and apoptosis in zebrafish embryos.

MS-222, the most widely used anaesthetic in fish, has been shown to induce embryotoxic effects in zebrafish. However, the underlying molecular effects are still elusive. This study aimed to investigate the effects of MS-222 exposure during early developmental stages by evaluating biochemical and molecular changes. Embryos were exposed to 50, 100 or 150 mg L-1 MS-222 for 20 min at one of three developmental stages (256-cell, 50% epiboly, or 1-4 somite stage) and oxidative-stress, cell proliferation and apoptosis-related parameters were determined at two time-points (8 and 26 hpf). Following exposure during the 256-cell stage, the biochemical redox balance was not affected. The genes associated with glutathione homeostasis (gstpi and gclc) were affected at 8 hpf, while genes associated with apoptosis (casp3a and casp6) and cellular proliferation (pcna) were found affected at 26 hpf. An inverted U-shaped response was observed at 8 hpf for catalase activity. After exposure at the 50% epiboly stage, the gclc gene associated with oxidative stress was found upregulated at 8 hpf, while gstpi was downregulated and casp6 was upregulated later on, coinciding with a decrease in glutathione peroxidase (GPx) activity and a non-monotonic elevation of protein carbonyls and casp3a. Additionally, MS-222 treated embryos showed a decrease in DCF-staining at 26 hpf. When exposure was performed at the 1-4 somite stage, a similar DCF-staining pattern was observed. The activity of GPx was also affected whereas RT-qPCR showed that caspase transcripts were dose-dependently increased (casp3a, casp6 and casp9). The pcna mRNA levels were also found to be upregulated while gclc was changed by MS-222. These results highlight the impact of MS-222 on zebrafish embryo development and its interference with the antioxidant, cell proliferation and cellular death systems by mechanisms still to be explained; however, the outcomes point to the Erk/Nrf2 signalling pathway as a target candidate.

Implementation of a functional endpoint to the zebrafish embryotoxicity test to evaluate craniofacial abnormalities.

The inclusion of a read-out to detect functional consequences of craniofacial alterations in the zebrafish embryotoxicity test will allow to evaluate these alterations which are difficult to assess morphologically, and to detect alterations in cranial nerves functions leading to impairment of jaw movements. In this study we have established an ingestion test in zebrafish larvae younger than 120 hpf. To overcome the challenge of evaluating larvae which still do not present independent feeding behaviour, we have tested the ability of 72, 96 or 102 hpf larvae to ingest food mixed with fluorescent microspheres under several conditions (dark/light, with/without shaking) to find the best experimental set-up for the test. We have included the investigation of two substances as potential positive controls: ketoconazole and tricaine. Ketoconazole 10 µM exposure during development produced significant embryotoxic effects including a characteristic craniofacial alteration pattern consisting in impaired development of brain, nasal cavity, mouth opening and jaw, as well as a significant decrease in food intake. Tricaine exposure at 380 µM during the food availability period significantly decreased the food intake. The method proposed will be a useful alternative tool to animal testing to detect compounds inducing adverse effects on craniofacial development.

MS-222 short exposure induces developmental and behavioural alterations in zebrafish embryos.

MS-222 has been widely used as an anaesthetic in fish, thus, raising the need to infer about its toxicological safety during development. In this study, MS-222 toxicity in zebrafish embryos was evaluated after a 20-min exposure at different stages of development. Embryos exposed during the 256-cell stage displayed an increase in mortality, associated with defective early developmental pathways. Following exposure during the 50% epiboly stage, an increase in mortality and abnormal cartilage development, as well as changes in noggin expression were observed. Locomotor deficits were detected and associated with changes in early signalling pathways through the involvement of noggin. When exposed at the 1-4 somites stage, zebrafish were phenotypically normal, although presenting changes in the expression pattern of developmental genes. These findings indicate a teratogenic impact, independent of sodium channels that should be taken into consideration when MS-222 toxicity is discussed.

Design and synthesis of a potent, highly selective, orally bioavailable, retinoic acid receptor alpha agonist.

A ligand-based virtual screening exercise examining likely bioactive conformations of AM 580 (2) and AGN 193836 (3) was used to identify the novel, less lipophilic RARα agonist 4-(3,5-dichloro-4-ethoxybenzamido)benzoic acid 5, which has good selectivity over the RARß, and RARγ receptors. Analysis of the medicinal chemistry parameters of the 3,5-substituents of derivatives of template 5 enabled us to design a class of drug-like molecules with lower intrinsic clearance and higher oral bioavailability which led to the novel RARα agonist 4-(3-chloro-4-ethoxy-5-isopropoxybenzamido)-2-methylbenzoic acid 56 that has high RARα potency and excellent selectivity versus RARß (2 orders of magnitude) and RARγ (4 orders of magnitude) at both the human and mouse RAR receptors with improved drug-like properties. This RARα specific agonist 56 has high oral bioavailability (>80%) in both mice and dogs with a good PK profile and was shown to be inactive in cytotoxicity and genotoxicity screens.

Dimethocaine, a synthetic cocaine derivative: studies on its in vitro metabolism catalyzed by P450s and NAT2.

Dimethocaine (DMC), a synthetic derivative of cocaine, is distributed and consumed as "new psychoactive substance" (NPS) without any safety testing at the forefront. It is mainly metabolized by N-acetylation, N-deethylation or hydroxylation. Therefore, the aim of the presented study was to determine the human NAT and P450 isozymes involved in this major metabolic steps, to measure the kinetics of the reactions, and to estimate the contribution on in vivo hepatic clearance. For these studies, cDNA-expressed NATs and P450s were used and formation of metabolites after incubation was measured using LC-MS or LC-MS(n). For N-acetylation, NAT2 could be shown to be the only isoform catalyzing the reaction in vitro hence assuming to be the only relevant enzyme for in vivo acetylation. Kinetic profiles of all P450 catalyzed metabolite formations followed classic Michaelis-Menten behavior with enzyme affinities (Km values) between 3.6 and 220 µM. Using the relative activity factor approach, the net clearances for deethylation of DMC were calculated to be 3% for P450 1A2, 1% for 2C19, <1% for 2D6, and 96% for 3A4. The net clearances for hydroxylation of DMC were calculated to be 32% for P450 1A2, 5% for 2C19, 51% for 2D6, and 12% for 3A4. Furthermore, these data were confirmed by chemical inhibition tests in human liver microsomes. As DMC is metabolized via two main steps and different P450 isoforms were involved in the hepatic clearance of DMC, a clinically relevant interaction with single P450 inhibitors should not be expected. However, a slow acetylation phenotype or inhibition of NAT2 could lead to decreased N-acetylation and hence leading to an increased risk of side effects caused by this arylamine.

Multilayer mounting enables long-term imaging of zebrafish development in a light sheet microscope.

Light sheet microscopy techniques, such as selective plane illumination microscopy (SPIM), are ideally suited for time-lapse imaging of developmental processes lasting several hours to a few days. The success of this promising technology has mainly been limited by the lack of suitable techniques for mounting fragile samples. Embedding zebrafish embryos in agarose, which is common in conventional confocal microscopy, has resulted in severe growth defects and unreliable results. In this study, we systematically quantified the viability and mobility of zebrafish embryos mounted under more suitable conditions. We found that tubes made of fluorinated ethylene propylene (FEP) filled with low concentrations of agarose or methylcellulose provided an optimal balance between sufficient confinement of the living embryo in a physiological environment over 3 days and optical clarity suitable for fluorescence imaging. We also compared the effect of different concentrations of Tricaine on the development of zebrafish and provide guidelines for its optimal use depending on the application. Our results will make light sheet microscopy techniques applicable to more fields of developmental biology, in particular the multiview long-term imaging of zebrafish embryos and other small organisms. Furthermore, the refinement of sample preparation for in toto and in vivo imaging will promote other emerging optical imaging techniques, such as optical projection tomography (OPT).

Comparison of the effects of four anaesthetics on haematological and blood biochemical profiles in pikeperch (Sander lucioperca L.).

OBJECTIVES: The objectives of the study were to compare the effects of Propiscin, 2-phenoxyethanol, clove oil and tricaine methane sulphonate (MS 222), anaesthetics frequently used in aquaculture. DESIGN: The haematological and biochemical blood profiles of pikeperch (Sander lucioperca L.) anesthetized with Propiscin (1.5 ml L-1), 2-phenoxyethanol (0.3 ml L-1), clove oil (33 mg L-1), MS 222 (150 mg L-1) and non-anesthetized control group were tested. Each tested group was divided into two subgroups, the first subgroup was sampled in anaesthesia 10 min after application of the anaesthetic and the second one live on 24h. RESULTS: The erythrocyte count and haematocrit was significantly decreased in 2-phenoxyethanol (24 h) compared with control group (CG). The mean corpuscular haemoglobin concentration was significantly increased in 2-phenoxyethanol (10 min), Propiscin (10 min and 24 h) compared to CG. The 2-phenoxyethanol (10 min and 24 h), MS 222 (24 h), clove oil (24 h), and Propiscin (10 min and 24 h) showed significantly lower leukocyte count compared with CG. The level of glucose was significantly (p<0.05) elevated with MS 222 (10 min) and clove oil (10 min) compared with CG. The 2-phenoxyethanol (10 min and 24 h), MS 222 (24 h), clove oil (24 h), and Propiscin (24 h) showed significantly lower (p<0.01) ammonia levels compared with CG. The triacylglycerols was significantly decreased (p<0.01) with Propiscin (10 min and 24 h), MS 222 (24 h), clove oil (24 h) and with 2-phenoxyethanol (24 h) compared with CG. After 24 hours MS 222 (24 h) and Propiscin (24 h) anaesthesia, fish showed significantly lower (p<0.01) concentration of inorganic phosphate compared with CG. CONCLUSIONS: On the basis of this experiment, it appears that clove oil was associated with the lowest effects in pikeperch and therefore would be recommended as an alternative to MS 222, while Propiscin and 2-phenoxyethanol are not suitable for manipulation with pikeperch in aquaculture.

Daily rhythms of toxicity and effectiveness of anesthetics (MS222 and eugenol) in zebrafish (Danio rerio).

Although the chronotoxicity of xenobiotics is relatively well known in mammals, the existence of daily rhythms of drug toxicity and effectiveness in fish has been neglected to date. The aim of this research was to investigate the influence of the time (middle of the light phase [ML] versus middle of the dark phase [MD]) of exposure to two anesthetic substances (MS-222 or clove oil) commonly used with fish on the median lethal concentration (LC(50)) and swimming activity of zebrafish (Danio rerio). To this end, adult zebrafish were kept under a 12 h:12 h light-dark (LD) cycle and exposed to different concentrations of the anesthetics for 15 min at ML or MD. LC(50) calculations were performed using the Spearman-Karber program, whereas swimming activity was video-recorded and analyzed with specialized software. Zebrafish exhibited a mostly diurnal activity pattern (77.9% of activity occurring during daytime). The acute toxicity and mortality caused by MS-222 and eugenol varied with the time of exposure. For MS-222, the LC(50) was 170.6 ± 7.4 mg/L in fish exposed at ML and 215.6 ± 3.9 mg/L at MD, whereas for eugenol the LC(50) was 70.3 ± 3.1 mg/L at ML and 104.9 ± 5.4 mg/L at MD. Exposure to sublethal concentrations of MS-222 and eugenol altered the swimming patterns of zebrafish in a different manner depending on the time of exposure. Thus, the time required for decreasing swimming activity during exposure to anesthetics was shorter at ML than at MD, whereas the recovery period was longer during the day. In conclusion, these results revealed that the toxicity and effectiveness of both anesthetic substances is highest during daytime, the active phase of fish, thus suggesting a link between the daily rhythms of behavior and toxicity.

Synthesis and biological evaluation of chromium bioorganometallics based on the antibiotic platensimycin lead structure.

The recent discovery of the natural product platensimycin as a new antibiotic lead structure has triggered the synthesis of numerous organic derivatives for structure-activity relationship studies. Herein, we describe the synthesis, characterization and biological evaluation of the first organometallic antibiotic inspired by platensimycin. Two bioorganometallic compounds containing (eta(6)-pentamethylbenzene)Cr(CO)(3) (2) and (eta(6)-benzene)Cr(CO)(3) (3), linked by an amide bond to the aromatic part of platensimycin, were synthesized. Their antibiotic activities were tested against B. subtilis 168 (Gram positive) and E. coli W3110 (Gram negative) bacterial strains. Both compounds were found to be inactive against E. coli but derivative 2 inhibits B. subtilis growth at a moderate MIC value of 0.15 mM. To test the intrinsic toxicity of chromium, several chromium salts along with {eta(6)-(3-pentamethylphenyl propionic acid)}Cr(CO)(3) (5) and {eta(6)-(3-phenyl propionic acid)}Cr(CO)(3) (6) were tested against both bacterial strains. No activity was observed against E. coli for any of the compounds; B. subtilis growth was not inhibited by Cr(NO(3))(3) and only very weakly by 5, K(2)Cr(2)O(7) and Na(2)CrO(4) at MIC values of 0.5, 0.68 and 1.24 mM, respectively. Compounds 2, 3, 5 and 4 (the pure organic analogue of 2) show similar cytotoxicity against HeLa, HepG2 and HT-29 mammalian cell lines. Furthermore, the cellular uptake and the intracellular distribution of compounds 2, 3 and Cr(NO(3))(3) in B. subtilis were studied using atomic absorption spectroscopy to gain insight in to the possible cellular targets. Compound 2 was found to be readily taken up and distributed almost equally among cytosol, cell debris and cell membrane in B. subtilis.

Ontogenetic changes in the toxicity and efficacy of the anaesthetic MS222 (tricaine methanesulfonate) in zebrafish (Danio rerio) larvae.

Median lethal (LC(50)) and effective (EC(50)) concentrations for 1-h and 24-h exposures to the anaesthetic MS222 (tricaine methanesulfonate) were determined for zebrafish Danio rerio larvae ranging in age from 3 days postfertilization (dpf) to 9 dpf. Cessation of heart beat was used as the indicator of death (LC(50)) while failure to respond to direct mechanical stimulation of the head region was taken as an indication of deep anaesthesia (EC(50)). 1-h LC(50)s, 1-h EC(50)s and 24-h EC(50)s all decreased gradually but significantly (all P<0.01) with age. Mean values for 1-h LC(50)s were 1633 mg L(-1) and 730 mg L(-1), respectively, for 3 dpf and 9 dpf larvae. Mean value for 1-h and 24-h EC(50)s were 106 mg L(-1) and 100 mg L(-1), respectively, at 3 dpf and 65 mg L(-1) and 31 mg L(-1), respectively, at 9 dpf. The gradual increase with age in sensitivity to the anaesthetic implied by these indicators is probably a reflection of ontogenetic changes in the activity of detoxification pathways. Mean values for the 24-h LC(50) also decreased significantly (P<0.001) with age, from 566 mg L(-1) at 3 dpf to 64 mg L(-1) at 9 dpf. However, unlike the other indicators, the decrease was not gradual but occurred in a step-like fashion with virtually all of the change occurring between 4 dpf and 7 dpf. This sharp increase in sensitivity coincides with the shift in the major site of systemic ionoregulatory activity from the skin to the gills. The implications of these ontogenetic changes in lethal and effective levels for researchers or others intending to use the anaesthetic with fish larvae are discussed.

Dithiocarbamates have a common toxic effect on zebrafish body axis formation.

We previously determined that the dithiocarbamate pesticide sodium metam (NaM) and its active ingredient methylisothiocyanate (MITC) were developmentally toxic causing notochord distortions in the zebrafish. In this study, developing zebrafish were exposed to isothiocyanates (ITCs), dithiocarbamates (DTCs) and several degradation products to determine the teratogenic relationship of these chemical classes at the molecular level. All dithiocarbamates tested elicited notochord distortions with notochord NOELs from <4 to 40 ppb, while none of the ITCs caused notochord distortions with the exception of MITC. Carbon disulfide (CS(2)), a common DTC degradate, also caused distortions at concentrations >200 times the DTCs. Whole mount in situ hybridization of developmental markers for collagen (collagen2a1), muscle (myoD), and body axis formation (no tail) was perturbed well after cessation of treatment with pyrolidine-DTC (PDTC), dimethyl-DTC (DMDTC), NaM, MITC, and CS(2). Therefore, distinct albeit related chemical classes share a common toxic effect on zebrafish notochord development. To test the responsiveness of the distortion to metal perturbation, five metal chelators and 2 metals were studied. The membrane permeable copper chelator neocuproine (NCu) was found to cause notochord distortions similar to DTC-related molecules. DMDTC and NCu treated animals were protected with copper, and collagen 2a1 and no tail gene expression patterns were identical to controls in these animals. PDTC, NaM, MITC, and CS(2) were not responsive to copper indicating that the chelation of metals is not the primary means by which these molecules elicit their developmental toxicity. Embryos treated with DMDTC, NaM, and NCu were rescued by adding triciaine (MS-222) which abolishes the spontaneous muscle contractions that begin at 18 hpf. In these animals, only collagen 2a1 expression showed a similar pattern to the other notochord distorting molecules. This indicates that the perturbation of no tail expression is in response to the muscle contractions distorting the notochord, while collagen 2a1 is associated with the impact of these molecules on much earlier developmental processes.

Pigmentation development, defects, and patterning in summer flounder (Paralichthys dentatus).

Flounders offer unique opportunities to study the cytological basis of vertebrate pigmentation. Individual skin pigment cells are clearly visible at hatching, and flounder ontogeny includes a dramatic shift in overall pigmentation (from symmetrical to asymmetrical) during metamorphosis. Moreover, several types of malpigmentation occur in hatchery populations; although much effort has gone into reducing the frequency of such defects, their etiology remains poorly understood, and they have rarely been described at the cellular level. In this paper, we use light and fluorescence microscopy to describe the cytological basis of normal developmental changes and of common types of malpigmentation. We then discuss the implications of these observations for underlying patterning mechanisms.

Selective cytotoxicity and telomere damage in leukemia cells using the telomerase inhibitor BIBR1532.

Telomerase represents an attractive target for a mechanism-based therapeutic approach because its activation has been associated with unlimited proliferation in most cancer cells. Recently, a nonnucleosidic small molecule inhibitor, BIBR1532 (2-[(E)-3-naphtalen-2-yl-but-2-enoylamino]-benzoic acid), has been identified that is highly selective for inhibition of telomerase, resulting in delayed growth arrest of tumor cells. Here we examined the effects of BIBR1532 in different leukemia cell lines as well as in primary cells from patients with acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL) in short-term culture assays. We observed a dose-dependent direct cytotoxicity in concentrations ranging from 30 to 80 microM. Interestingly, cell death was not dependent on the catalytic activity of telomerase but was delayed in cells with very long telomeres. We observed time-dependent individual telomere erosion, which was associated with loss of telomeric repeat binding factor 2 (TRF2) and increased phosphorylation of p53. Importantly, the proliferative capacity of normal CD34(+) cells from cord blood and leukapheresis samples was not affected by treatment with BIBR1532. We conclude that using this class of telomerase inhibitor at higher concentrations exerts a direct cytotoxic effect on malignant cells of the hematopoietic system, which appears to derive from direct damage of the structure of individual telomeres and must be dissected from telomerase-suppressed overall telomere shortening.

Muscle weakness during tricaine anesthesia.

An isolated preparation of tadpole tail muscle was used to assess the peripheral effects of tricaine (3-aminobenzoic acid ethyl ester) at anesthetic concentrations and under physiological conditions. The drug effect on the electrically-evoked twitch was tested using short-pulse durations that elicited synaptically mediated effects or longer-duration pulses that stimulated the muscle directly. Tricaine reduced both types of response anesthetic and even subanesthetic concentrations. At steady state concentrations that produced surgical anesthesia in vivo, tricaine reduced the directly evoked response by about half. It is concluded that tricaine anesthesia has a pronounced peripheral effect on neuromuscular function and that direct effect(s) on muscle are a major component.

Discriminative stimulus effects of esteratic local anesthetics in squirrel monkeys.

A number of esteratic local anesthetics serve as positive reinforcers and produce cocaine-like discriminative stimulus effects in animals. It has been suggested that the affinity of these compounds for a site on the dopamine transporter, and not their local anesthetic actions, is responsible for these abuse-related behavioral effects. In the present study, three local anesthetics previously shown to be self-administered in animals were examined in squirrel monkeys trained to discriminate cocaine (0.3 mg/kg) from saline in a two-lever, food-reinforced procedure. Dimethocaine (0.1-3.0 mg/kg) fully and dose-dependently substituted for cocaine. Doses of dimethocaine (1.7 mg/kg) and cocaine (0.3 mg/kg) which produced full (> 80%) substitution for cocaine were administered in combination with the dopamine D1 receptor antagonist SCH 39166 ((-)-trans-6,7,7a,8,9,13b-hexahydro-3-chloro-2-hydroxy-N-methyl-5H -benzo [d]naphtho-(2,1-b)azepine) and the dopamine D2 receptor antagonist raclopride (both at 0.003-0.03 mg/kg). SCH 39166 fully blocked the cocaine-like discriminative stimulus effects of dimethocaine and cocaine, but raclopride produced only partial antagonism of cocaine-lever selection. In addition, there was some evidence that raclopride blocked cocaine-lever responding produced by a lower dose of dimethocaine. In substitution studies, neither procaine (1-10 mg/kg) nor chloroprocaine (1-30 mg/kg) produced cocaine-like effects. These results support a role for dopamine in the behavioral effects of some local anesthetics.

The acute toxicity and in vivo antitumour activity against L1210 leukemia of triphenyltin 3,5-diisopropylsalicylate, bis (di-n-butyl-(s)-2-pyrrolidone-5-carboxylato)tin oxide and di-n-butyltin bis(3-amino-4-methylbenzoate).

Triphenyltin 3,5-di-isopropylsalicylate, compound 1, is characterized by a maximum tolerated dose (MTD) of 20 mg/kg. Bis[di-n-butyl(2-pyrrolidone-5-carboxylato)tin] oxide, compound 2, and (di-n-butyltin bis(3-amino-4-methyl-benzoate), compound 3, exhibit similar acute toxicities (MTD = 8 mg/kg) despite their lower in vitro activity, as compared to compound 1, against the two human tumor cell lines MCF-7 and WiDr. All three are inactive in vivo against L1210 leukemia in mice.