High Concentrations Of Cocaine Research Articles

B-319 Drugs of Abuse: Correlation of Immunoassay Absorbance With Concentration By LC-MS/MS and False Negative Rates on the Roche Analyzer

Abstract A common practice for drug abuse testing is to first screen using immunoassay (IA) then confirm using tandem mass spectrometry (LC-MS/MS) if the screening result is indeed positive. Immunoassays are qualitative and a positive result is based on an absorbance reading over a cutoff value specified by the manufacturer. Despite that, one method of investigating unexpected results is to check for an absorbance close to the cutoff value, as that may indicate an analyte concentration close to the limit of detection and the need for further investigation. Here we investigate the validity of this approach regarding potential false positives/false negatives by using quantitative LC-MS/MS assays. These explicitly model instrument response as a function of concentration and are expected to be more reliable than absorbance. This abstract aims to answer two questions: Is there a relationship between absorbance by IA and concentration of analyte as measured by LC-MS/MS that can be used in clinical interpretations? What is the false negative rate (FNR) by IA screening on the Roche automated analyzer? We collected discarded patient urine samples that were screened for amphetamines (AMP), cocaine (COC) and THC to get a total of 10 negative and 10 positive samples of each analyte with a range of absorbance values, as well as preliminary data for benzodiazepines, opiates, and oxycodone. All samples were tested again 5 times in one day using a Roche Cobas c501 chemistry analyzer. The cutoff concentrations for the immunoassays are 500 ng/mL for AMP, 300 ng/mL for COC and 50 ng/mL for THC. In-house LC-MS/MS assays gave quantitative measurements with a limit of quantitation of 30 ng/mL for AMP and COC and 10 ng/mL for THC. The results obtained from LC-MS/MS were considered as reference values to determine the FNR of the IAs. The linear regression between absorbance and concentration was calculated in Excel using the average absorbance of each sample. The amphetamine IA results showed a concordance with LC-MS/MS of 80%, a FN rate of 20%, and an R2 of 0.80 between absorbance and concentration. The cocaine IA results showed a concordance with LC-MS/MS of 65%, a FN rate of 41%, and an R2 of 0.24. The THC IA results showed a concordance with LC-MS/MS of 95%, a FN rate of 9%, and an R2 of 0.94. The amphetamine IA shows moderate agreement with LC-MS/MS and accurately detected 80% of the positive specimens. The absorbance and concentration show good fit to the regression model. The cocaine IA shows fair agreement with LC-MS/MS and has an LOD too high to detect the presence of drug in most specimens that were found positive by LC-MS/MS. The lack of fit to the regression model is due to specimens with very high concentrations of COC that do not cause a proportional increase in absorbance. The THC IA shows excellent agreement with LC-MS/MS and excellent fit to the regression model. These findings indicate the use of absorbance may be able to assist in test interpretation for specimens near the cutoff value.

Characterization and forensic identification of a novel cocaine charcoal smuggling matrix

Every year new and sophisticated drug-smuggling methods are encountered by law enforcement agencies all over the globe. A seized cross-border smuggling attempt was found to contain charcoal. This unexpected smuggling cargo raised doubts as to the real content of the material. Initial testing indicated that the black charcoal-like material contained cocaine. The material was forensically tested for cocaine presence and quantitatively analyzed for cocaine concentration. Initial testing with an illicit substance identification field kit and FTIR revealed that some of the material contained cocaine whereas other pieces were completely free of cocaine. Cocaine-containing material was quantitatively measured and was found to consist of over 50% cocaine. In addition, the morphology and element composition of the suspected matrix were analyzed by SEM and EDX. The results pointed to some structural and composition differences between material loaded with cocaine and charcoal free of the drug. One of the most significant and surprising differences in the element composition measurements was the finding of iron in the cocaine-containing material. Moreover, thermogravimetric analysis was performed on the samples to support the material composition analyses. Although the exhibits did not display a homogenous presence and concentration of cocaine, the porous morphological structure and the high cocaine concentration in some of the samples reveal that the carbon-rich matrix has a potential for high capacity drug uptake. The iron accompanying the cocaine is probably a left over product of the cocaine masking process.

Seasonal monitoring of cocaine and benzoylecgonine in a subtropical coastal zone (Santos Bay, Brazil).

Illicit drugs and their metabolites represent a new class of emerging contaminants. These substances are continuously discharged into wastewater which have been detected in the aquatic environment in concentrations ranging from ng.L-1 to μg.L-1. Our study detected the occurrence of cocaine (COC) and benzoylecgonine (BE) in a subtropical coastal zone (Santos Bay, SP, Brazil) within one year. Water samples (surface and bottom) were collected from the Santos Submarine Sewage Outfall (SSOS) area. COC and BE were measured in the samples using ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-ESI-MS/MS). Concentrations ranged from 12.18 to 203.6 ng.L-1 (COC) and 8.20 to 38.59 ng.L-1 (BE). Higher concentrations of COC were observed during the end of spring, following the population increase at summer season. COC and its metabolite occurrence in this coastal zone represent a threat to coastal organisms.

Discrimination between chewing of coca leaves or drinking of coca tea and smoking of “paco” (coca paste) by hair analysis. A preliminary study of possibilities and limitations

BackgroundHair analysis is a suitable way to discriminate between coca chewers and consumers of manufactured cocaine using the coca alkaloids hygrine (HYG) and cuscohygrine (CUS) as markers. In the present preliminary study it was examined whether CUS and HYG can be detected in hair of occasional and moderate coca chewers or coca tea drinkers, whether CUS and HYG appear in hair of PACO consumers (smoking coca paste waste), and whether anhydroecgonine methyl ester (AEME) is a useful cocaine smoking marker in this context. MethodThree groups were included: 10 volunteers from Buenos Aires with occasional or moderate chewing of coca leaves or drinking coca tea, 20 Argentinean PACO smokers and 8 German cocaine users. The hair samples (1–4 segments) were analyzed by a validated LC–MS/MS method for cocaine (COC), norcocaine (NC), benzoylecgonine (BE), ecgonine methyl ester (EME), cocaethylene (CE), cinnamoylcocaine (CIN), tropacocaine (TRO), AEME, CUS and HYG. For comparison, eight samples of coca leaves or coca tea were analyzed. ResultsOnly low concentrations of COC were found in hair of seven occasional users of coca leaves or coca tea (0.010–0.051 ng/mg). For three moderate chewers of coca leaves all compounds were detected including AEME but except TRO. The hair samples of PACO smokers contained much higher concentrations of COC (0.027–341 ng/mg, mean 37.4 ng/mg) and its metabolites. CUS was not found in these samples but traces of HYG were seen in 8 of 37 hair segments. AEME as a marker for coca smoking was detected in hair of 15 smokers. In comparison to COC, the concentrations of EME and CIN were higher for PACO smokers than for German cocaine consumers. AEME (56 ± 20 μg/g) was detected in all coca leave and coca tea samples which explains the detection of this substance in hair of coca chewers. Therefore, its use for differentiation between coca chewers and PACO smokers is limited. ConclusionCUS remains to be the most suitable marker in hair for chewing coca leaves or drinking coca tea more frequently than two times per month since it does not appear in hair of Argentinean PACO smokers and German cocaine users. Contrary to a previous proposal, the ratios CIN/COC and EME/COC appeared not to be applicable as criteria for this purpose because of the higher concentration of these alkaloids in hair of PACO smokers. More research is needed to assess the value of AEME in hair of South American coca leave or cocaine users.

Screening for cocaine on Euro banknotes by a highly sensitive enzyme immunoassay

This study focused on quantitative detection of cocaine on Euro banknotes in Germany. A sensitive direct competitive immunoassay was developed and optimized with a limit of detection (LOD) of 5.6ng/L. Exhaustive cocaine extraction by solvent was tested using different methanol concentrations and buffered solutions. Cross-reactivity studies were performed to determine the degree of interference of cocaine metabolites with the immunoassay. Sixty-five Euro banknotes obtained from different districts in Berlin were evaluated. A 100% contamination frequency with cocaine was detected. A comparison between the amount of cocaine extracted by cotton swabbing of one square centimeter of the banknote showed a good correlation for lower contamination levels. This assay showed high sensitivity of detecting pg of cocaine per 1cm2 of one banknote by swabbing 1cm2: 0, 14, and 21pg/cm2. Moreover, three notes of different denominations revealed high cocaine concentration; 1.1mg/note, and twice 55µg/note.

Increased MFG-E8 expression and its implications in the vascular pathophysiology of cocaine abuse.

The aim of this study was to examine the possible involvement of smooth muscle cell remodeling and the induction of MFG-E8 (milk fat globule protein epidermal growth factor-VIII) in vascular pathophysiology during cocaine administration in cultured cells and rats. Cocaine exerts bifurcate effects on vascular cells; it stimulates vasoconstriction through enhancement of catecholamine release at low doses, while it suppresses cardiovascular functions through inhibition of ion channels at high doses. Short-term exposure to a high concentration of cocaine (3 mM, 24 hr) resulted in cell death of A7r5 rat aorta-derived smooth muscle cells. On the other hand, long-term exposure of the same cells to a low concentration (0.3 mM, ~7 days) resulted in a transient increase in MFG-E8 expression followed by an increased tendency toward cyclin D1, PCNA (proliferating cell nuclear antigen), and CDK4 (cyclin-dependent protein kinase-4) expression. Interestingly, autophagy was not induced, but rather was impaired, in cocaine-treated cells. Increased expressions of MFG-E8, PCNA, and CDK4 were also observed in the aortic vascular cells of rats administered cocaine (50 mg/kg, 2 days, i.v.), confirming that cocaine induced MFG-E8 expression in vivo. Taken together, the results show that MFG-E8 is induced in vascular cells exposed to cocaine, and that this induction is likely to be involved in the vascular toxicity elicited by cocaine abuse.

Fatal poisoning with cocaine and tramadol in a former professional cyclist

We describe a case of an acute overdose in a former professional cyclist with a heavy past of doping and drug addiction. A 31-years-old man was admitted in hospital for generalized tonico-clonic seizures concomitant with hyperthermic syndrome and unstable hemodynamic status. During his transport to the emergency department, he presents two cardiopulmonary arrest, which required an external electric shock and adrenaline administration. History of the disease revealed that patient is in a context of benzodiazepines withdrawal. He complained for five days about shivers, sweats, headaches and nausea with change of his general state. Moreover, he would have taken cocaine at night previous his admission. During his coverage by emergency service, the body temperature = 40 °C, SpO2 = 70%, blood pressure = 70/40 mmHg and heart rate = 70/min. Several cardiac, biological and toxicological examinations were implemented during the hospitalization. Toxicology analysis was realized on blood. Blood screening was realized by LC-MS/MS, after liquid/liquid extraction in acidic and basic conditions. Specific methods were done for cocaine and tramadol quantification by LC-MS/MS, and for lidocaine by GC-MS. Other identified substances were quantified by LC-MS/MS or immunoassay analyses. The electrocardiogram highlights conduction abnormalities (slow QT then ventricular extrasystoles). Biological examination revealed a mix severe acidosis (pH = 7.27, paCO = 6.64 kPa, lactates = 5.4 mmol/L), an hypokaliemia (2.2 mmol/L), and an acute kidney failure with creatinine at 169 μmol/L associated with rhabdmyolyse (CPK = 13,000 U/L). Toxicological research revealed the presence in blood of high concentration of cocaine and its metabolites (cocaine concentration = 290 ng/mL, benzoylecgonine concentration = 3000 ng/mL). Moreover, several medicine drugs were also detected in blood sample (tramadol, acetaminophen, lidocaine and hydroxyzine). All are in therapeutic concentrations excepted tramadol, which was measured at the concentration of 1880 ng/mL (and 2000 ng/mL for O-DMT, its active metabolite). This fatal overdose with a massive cocaine consumption associated with a large dose of tramadol presented a mixt adrenergic-serotoninergic syndrome with seizures, hyperthermia, blood pressure fluctuations, acidosis and hypokaliemia and rhabdomyolysis. Let us note that the high concentration of tramadol found in this case, has probably potentiate the toxic effect of cocaine. Interestingly, the main part of the toxic symptoms observed linked to tramadol seem rather in touch with the inhibition of the recapture of monoamine than with the opioid activity leading to mixt adrenergic-serotoninergic syndrome [1]. This ascendancy of serotoninergic syndrome may be related with drug-abusing profile of the patient.

The Effect of Cocaine on C6 Glial Cells and its Modulation of Oxygen Consumption, Growth and Transcription Factors

Cocaine is a widely abused and addictive psychostimulant drug that acts on the central nervous system (CNS). Glial cells may be the first to experience the toxic effects of cocaine in the mammalian brain, since glial cells outnumber neurons by 10–1. Glial cells were grown in 10% FBS (fetal bovine serum) in Dulbecco's modified Eagle medium (DMEM) and penicillin/streptomycin (100 Units/0.1 mg per ml). Treatment of glial cells with cocaine for 24 and 48 hours caused significant loss of cell viability. We investigated the expression of Hypoxia Inducible Factor‐1α (HIF‐1α) and Vascular Endothelial Growth Factor (VEGF) in 1, 2, 3 and 4 mM of cocaine exposure and also in control samples of glial cells. Preliminary data from different studies indicated that hypoxia is a potent inducer of VEGF in vitro partly because of an increased transcription rate, mediated by binding of HIF‐1 α to a hypoxia responsive element, VEGF gene. Thus following cocaine treatment of various concentrations mentioned above and RNA isolation, real time PCR (Polymerase Chain Reaction ) was carried out using the IQ Sybr Green Super mix. Expression of HIF‐1 α followed by VEGF observed in each concentration compared to control and Flow Cytometry and Elisa experiments confirmed the expression. Oxygen consumption study showed significant reduction in higher concentrations of cocaine. We concluded that cocaine may cause hypoxia in rat C6 glial cells via expression of vascular endothelial growth factor (VEGF).Acknowledgment: This research was supported by National Institute on Minority Health Disparities of the National Institute of Health under award number G12MD007582,FAMU RCMI, Pharmaceutical Research Center

Differential Sensitivity of A2A and Especially D2 Receptor Trafficking to Cocaine Compared with Lipid Rafts in Cotransfected CHO Cell Lines. Novel Actions of Cocaine Independent of the DA Transporter

The effects of low and high concentrations of cocaine have been studied in vitro on the trafficking of plasma membrane A(2A) and D(2) immunoreactivities in previously characterized A(2A)-D(2) CHO cell lines. Receptor double immunofluorescence staining was performed with D(2) and A(2A) antibodies, planar lipid rafts immunolabeling with biotinylated cholera toxin subunit B and membrane invaginations with an anti-caveolin-1 antibody. A computer-assisted image analysis demonstrated a substantial and highly significant rise of membrane-associated D(2) immunoreactivity (IR) after 8 h of exposure to a low concentration of cocaine (150 nM). At this low concentration of cocaine, there was also an increase of membrane associated A(2A) immunoreactivity but smaller and less significant. However, this increase became considerably larger and highly significant at 150 microM at which concentration the rise of D(2) immunoreactivity had begun to disappear. It may be suggested that an allosteric action of cocaine at 150 nM on the D(2) receptors may primarily increase the insertion of D(2) monomers, homomers and also of a subpopulation of A(2A)-D(2) heteromers from the cytoplasm into the plasma membrane due to the conformational change induced by cocaine in the D(2) receptor. The planar lipid rafts and the caveolae are only affected by the higher concentrations of cocaine. It is proposed that changes in D(2) and A(2A)-D(2) trafficking induced by allosteric actions of cocaine at D(2) receptors may contribute to the alterations of D(2) signaling found in cocaine abusers.

Crack cocaine causing fatal vasoconstriction of the aorta

Cocaine is the most powerful central nervous stimulant found in nature. All forms of cocaine cause tachycardia and vasoconstriction. A smokable, rapidly reacting form of cocaine base, “crack,” is highly addictive. Smoking crack introduces a high concentration of cocaine into the bloodstream, rendering it especially dangerous. We report a case that visually demonstrates severe aortic vasoconstriction from the suprarenal aorta and extending to both femoral arteries and beyond, resulting in renal failure and fatal bowel ischemia after a 5-day binge of crack cocaine.

Cocaine Induces a Differential Dose‐Dependent Alteration in the Expression Profile of Immediate Early Genes, Transcription Factors, and Caspases in PC12 Cells

Cocaine is a widely used drug of abuse and psychostimulant that acts on the central nervous system by blocking the dopamine reuptake sites. PC12 cells, a rat pheochromocytoma clonal line, in the presence of nerve growth factor (NGF), multiply and differentiate into competent neurons that can synthesize, store, and secrete the neurotransmitter dopamine (DA). In the present study, we evaluated the effect of increasing doses of cocaine on the expression of immediate early genes (IEGs), c‐fos and c‐jun, and closely related transcription factors, SP‐1 and NF‐kβ, at 24 h after the exposure to cocaine (50, 100, 200, 500, 1000, 2500 μM) in NGF‐differentiated PC12 cells. Cocaine (50‐500 μM) resulted in significant induction of the expression of c‐fos, c‐jun, SP‐1, and NF‐kβ. However, higher concentrations of cocaine (1000 and 2500 μM) resulted in the downregulation of these expressions after 24 h. To further understand the role of dose‐dependent changes in the mechanisms of cell death, we evaluated the protein expression of apoptotic markers. A concentration‐dependent increase in the expression of caspase‐9 and ‐3 was observed up to 500 μM cocaine. However, the higher dose did not show any expression. We also evaluated the effect of increasing doses of cocaine on DA concentration and the expression of dopamine transporter (DAT). A significant dose‐dependent decrease in the concentration of DA as well as the expression of DAT was observed 24 h after the exposure of PC12 cells to cocaine. Therefore, in the present study, we reported that cocaine has both upstream and downstream regulatory actions on some IEGs and transcription factors that can regulate the mechanism of cell death, and these effects on gene expression are independent of its action on the dopaminergic system.

Intracranial Self-Administration of Cocaine within the Posterior Ventral Tegmental Area of Wistar Rats: Evidence for Involvement of Serotonin-3 Receptors and Dopamine Neurons

The rewarding properties of cocaine have been postulated to be regulated, in part, by the mesolimbic dopamine (DA) system. The present study assessed whether adult female Wistar rats would self-administer cocaine directly into the ventral tegmental area (VTA). Following guide cannulae surgery aimed at either the posterior or anterior VTA, subjects were placed in an operant box equipped with an active lever that caused the delivery of the infusate and an inactive lever that did not. Posterior and anterior VTA subjects were randomly assigned to one of six groups that self-administered either artificial cerebrospinal fluid (aCSF) or 25 to 400 pmol cocaine/100 nl in aCSF for the first four sessions, aCSF in sessions 5 and 6, and the acquisition dose of infusate during session 7. Additionally, the effects of increasing the time-out period, higher concentrations of cocaine, coadministration of a 5HT3 antagonist, and coadministration of a D2/3 agonist on self-infusion of cocaine were determined. Self-infusions were maintained when the time-out period was extended from 5 to 25 s. Coinfusion of a 5HT3 antagonist or D2/3 agonist blocked the self-infusion of cocaine. In contrast, rats did not self-administer 25 to 400 pmol/100 nl cocaine into the anterior VTA. Additionally, rats did not self-administer either 800 or 1600 pmol/100 nl cocaine into the posterior or anterior VTA. Overall, the data indicate that the VTA is functionally heterogeneous with regard to the rewarding actions of cocaine and that the reinforcing effects of cocaine within the posterior VTA are mediated by activation 5-HT3 receptors and DA neurons.

Removing and identifying drug contamination in the analysis of human hair

The procedure used in this laboratory for removing and identifying contamination of hair specimens with drugs is demonstrated by its application to hair contaminated by various experimental models. The models include soaking; coating with drug followed by sweat conditions for 6 h; and soaking in a very high concentration of cocaine followed by storage and multiple shampoo treatments. A multi-part wash procedure along with a wash criterion is applied to all samples containing drug above the cutoff. The failure of the wash criterion is a signal that the sample may be positive due to contamination rather than use, and in the absence of other over-riding evidence, the sample would be considered to be negative for drug use. This Wash Criterion has also been tested with hair from subjects demonstrated to be drug users by one or more drug-positive urines; in these studies, all hair samples from demonstrated users passed the Wash Criterion test.

Bodypacking--an increasing problem in The Netherlands: conservative or surgical treatment?

Evaluation of diagnostic work-up and treatment of bodypackers. Identification of predictive factors for surgical treatment. Retrospective descriptive study. Teaching hospital, The Netherlands. All 40 consecutive patients, admitted during the period 1995-99 inclusive to the surgical department with the diagnosis of "bodypacker". Age, sex, medical history, additional diagnostic investigation, treatment, morbidity, and mortality. There were 36 men and 4 women, the reason for whose visit to the emergency department was the persistence of cocaine packages in the body. 24 had abdominal pain and 15 patients were vomiting. In all cases packages were identified on an abdominal radiograph taken supine. The cocaine concentration in the urine was raised in 8/13 patients. During admission 18/40 patients (45%) were operated on, and in 9 patients (23%) a previously unsuspected lesion was found, which was causing the obstruction. Factors predictive of the need for surgical treatment were: abdominal history (p = 0.01), abdominal pain while in the emergency department (p = 0.001), location of the packages of cocaine in the stomach and small intestine on the abdominal radiograph (p = 0.05), and a high concentration of cocaine in the urine (p = 0.03). 9 of the patients operated on developed complications; no patient died. The number of bodypackers admitted to the surgical department increases. More than half the patients could be treated conservatively. Factors predictive of the need for surgery are: abdominal history, pain, high obstruction, and cocaine in the urine. In 9 of the patients operated on an unsuspected disease was found that was causing the obstruction.

Maternal and Transplacental Effects of Cocaine.

Pregnancy is a dynamic process, and maternal as well as fetal risks from cocaine use in pregnancy may differ as pregnancy progresses. Three areas of biology offer opportunities for reevaluating cocaine's effects in pregnancy: (1) Maternal cardiovascular and neurologic responses to cocaine hydrochloride are enhanced when compared with responses in nonpregnant subjects to the same dose per kilogram or to metabolites of crack cocaine. (2) During first trimester placental implantation, oxygen availability to the fetus may normally be limited. Cocaine-induced uterine artery vasoconstriction may lead to reperfusion and oxygen toxicity to the fetus from released reactive oxygen species. (3) Cocaine transport in the first and early second trimester may, in part, be across the placental chorion-amnion. Lacking a skin barrier, the fetus at mid-pregnancy may come in direct contact with high concentrations of cocaine in amniotic fluid, a reservoir that clears cocaine slowly, thereby prolonging exposure during critical periods of fetal neurotransmitter formation. Exploring these three areas of biology may offer new approaches to understanding the ultimate impact of prenatal cocaine exposure on maternal and fetal biology.

Cocaine Inhibits Chloride Secretion by Swine Tracheal Submucosal Gland Cells Grown in Culture

Active ion transport by airway submucosal gland cells plays an important role in maintaining effective mucociliary clearance. Alterations in the clearance will affect normal airway functions. Nasal and airway mucosal surfaces are exposed to high concentrations of cocaine after snorting crystals or smoking the freebase form, causing respiratory complications. The actions of cocaine on airway gland cell ion transport were determined by measuring the basal electrical properties and ion transport activated by acetylcholine (ACh) and isoproterenol (ISO) in swine tracheal submucosal gland cells grown in culture. The cells were enzymatically isolated, purified on Percoll density gradients, and then grown on microporous inserts at an air interface. Chloride secretion was measured as short-circuit current (Isc) utilizing Ussing chamber methodology. Amiloride (10 μm) was added mucosally to block sodium reabsorption. The maximal changes in Isc (ΔIsc) induced by ACh and ISO were 42.0 ± 4.4 and 8.8 ± 0.8 μA/cm2. Cocaine decreased basal Isc and the increase in Isc induced by ACh and ISO. The concentration–response relationships for ACh and ISO were shifted to the right by cocaine (1–3 mm) when applied serosally. The maximal increase in Isc induced by ISO was inhibited by serosal cocaine, but, the maximal response to ACh was not. Thus, cocaine inhibits ACh-induced increases in Isc by competitively blocking the receptor and the response to ISO by noncompetitive inhibition. Mucosal cocaine reduced the maximal response to ACh and ISO and also caused a rightward shift. We conclude that cocaine directly alters submucosal gland cell function by reducing the basal and stimulated ability to transport ions and fluid.

Effects of cocaine and cocaine metabolites on mouse development in vitro

The use of cocaine use has been associated with adverse developmental effects in humans, and cocaine administration produces developmental toxicity in animal models. However, whether the adverse effects produced during organogenesis are due directly to the effects of cocaine or its metabolites remains to be established. This study was therefore undertaken to compare the morphological effects of cocaine and its metabolites, ecgonine, benzoylecgonine (BE) and ecgonine methyl ester (EME) in whole embryo culture (WEC) using early somite stage ICR mice. Cocaine produced a concentration-dependent induction of defects including effects on craniofacial development such as neural tube closure defects (NTDs). Concentrations of cocaine of 51.4 μ m or more produced dysmorphogenesis and 100% of the embryos exhibited NTDs at 441 μ m. EME also induced defects at concentrations of 400 μ m or above. Neither ecgonine nor BE altered embryogenesis at concentrations of 2000 μ m or less. The incidence of cocaine-induced NTDs was dependent on the length of exposure to cocaine. At 294 μ m, exposures of 3 hr or more were required to alter development when evaluated at the end of a 24-hr culture period. Lower cocaine concentrations required longer exposure periods (6 or 12 hr) to produce dysmorphogenesis. The incidence of NTDs appears to follow the area under the concentration time curve and is not solely dependent on the peak cocaine concentration in the medium. Exposure of conceptuses to a combination of cocaine and EME produced a high incidence of NTDs. These results suggest that the concentration of cocaine or EME required to induce NTDs in vitro is higher than the teratogenic concentration in vivo. Additionally, the time required for high concentrations of cocaine to induce NTDs is longer than the serum half-life of cocaine reported in vivo following a single administration. Thus, NTDs produced by cocaine administration appear not to be due solely to the effect of cocaine or its metabolites on the conceptus but may involve effects on extraembryonic and/or maternal tissues as well.

Biomass accumulation and alkaloid content in leaves of Erythroxylum coca and Erythroxylum novogranatense var. novogranatense grown in soil with varying pH

Summary Erythroxylum coca var. coca Lam. ( E. coca ) and Erythroxylum novogranatense var. novogranatense (Morris) Hieron ( E. novo, novogranatense ) were grown in greenhouse pots of Monmouth sandy loam, over a pH range of 3.5 through 7.0, to determine patterns for biomass accumulation and the content of hygrine, tropinone, methyl ecgonine, cuscohygrine, tropacocaine, cocaine, cis - and trans -cinnamoylcocaine in their leaves. Optimal growth for E. coca occurred at pH 3.5, and for E. novo. novogranatense at pH 4.7 through 6.0. The pH extreme (sub-optimal biomass accumulation, above and below optimal pH) for E. coca was ≥6.5 and for E. novo, novogranatense ≤ 3.5 and ≥ 6.5. Hence, E. coca is tentatively termed a calcifuge (Al-tolerant, Fe-inefficient) while E. novo. novogranatense more nearly resembles a calcicole (Fe-efficient, Alsensitive). Increases in leaf alkaloid content observed at the pH extremes for E. coca and E. novo, novogranatense appeared to be caused by plant stress. E. novo. novogranatense was more tolerant to the high pH extreme than E. coca . Twenty-two months after transplanting, 55% of E. coca plants grown at soil pH 7.0 died, and those that remained were chlorotic. In addition, among E. coca plants grown at soil pH 6.5, only 55 % were alive at the third harvest. Cocaine concentration in E. coca was independent of leaf biomass. Leaves of E. coca grown in soil at pH 3.5 through 6.0 had higher concentrations of cocaine than those of E. novo. novogranatense . Conversely, leaves of E. novo. novogranatense contained a higher concentration of trans - and cis -cinnamoylcocaine and methyl ecgonine than those of E. coca . The least abundant alkaloid observed in E. coca and E. novo. novogranatense was tropinone which was low or commonly not detected. Of the eight alkaloids monitored in leaves of E. novo. novogranatense grown in soils at various pH levels, methyl ecgonine was the most abundant.

* - The effects of in vitro cocaine exposure on human sperm motility, intracellular calcium, and oocyte penetration

To determine if cocaine exposure affects human sperm motility, intracellular calcium level, and fertilizing capability. Human semen samples were treated with 1 to 1,000 microM cocaine hydrochloride for up to 2 hours in vitro. Sperm motion kinematics were measured by computer-assisted semen analysis (CASA). Spermatozoan intracellular calcium was determined by laser cytometry. The sperm fertilizing capability was assessed using the zona-free hamster oocyte penetration test. After a short exposure (15 minutes) to cocaine, the sperm motion kinematic parameters, straight line velocity and linearity, were decreased in the high concentration groups. However, after a longer exposure (2 hours) to cocaine, the differences were no longer significant. Cocaine treatment did not alter spermatozoa intracellular calcium levels. Most importantly, human sperm treated with cocaine at a high concentration were fully capable of penetrating zona-free hamster oocytes. Human spermatozoa acutely exposed to high concentrations of cocaine initially demonstrate a decrease in two motion kinematics, straight line velocity and linearity. However, overall, cocaine exposure had no significant effects on sperm motility and fertilizing capability.

Time Course of Direct Cardiotoxic Effects of High Cocaine Concentration in Isolated Rabbit Heart

Studies of acute cocaine cardiotoxicity are often confounded by the effects of cocaine on peripheral hemodynamics and an intact sympathetic nervous system. To study the direct effects of an acute dose of cocaine on heart, we used Langendorff-perfused isolated rabbit hearts. The hearts were attached to a Langendorff perfusion system through the aorta and were perfused with Krebs-Henseleit buffer. In nonpaced hearts, cocaine in concentrations of 10, 25, and 50 microM (reported to be in the range of drug concentrations lethal for humans) caused dose-dependent deterioration in heart contractility (decrease in +dP/dt, -dP/dt, and developed pressure) and coronary flow, when measured at 10, 20, and 30 min of the protocol. Cocaine 25 and 50 microM also caused a decrease in heart rate (HR). In paced hearts treated with cocaine 50 microM, early events included a progressive decrease in +dP/dt (by 15% vs. baseline at 20 s of perfusion and by 26% vs. baseline and 19% vs. control group at 30 s of perfusion; p < 0.05) and in -dP/dt (by 20% vs. baseline at 30 s of perfusion and by 25% vs. baseline and 15% vs. control group at 40 s of perfusion, p < 0.05). Decrease in HR (failure to pace) was observed only at 60 s of perfusion and averaged 20% versus both baseline and control group (p < 0.05). No changes in coronary flow were observed during the first 60 s after onset of cocaine administration.(ABSTRACT TRUNCATED AT 250 WORDS)