Indiana University-Purdue University Indianapolis (IUPUI) / Environmental exposure to lead (Pb) and cannabis use are two of the largest public health issues facing modern society in the United States and around the world. Exposure to Pb in early life has been unequivocally shown to have negative impacts on development, and recent research is mounting showing that it may also predispose individuals for risk of developing substance use disorders (SUD). At the same time, societal and legal attitudes towards cannabis (main psychoactive component delta-9-tetrahydrocannabinol) have been shifting, and many American states have legalized the recreational use of cannabis. It is also the 3rd most widely used drug of abuse in the US, and rates of cannabis use disorder are on the rise. This thesis sets out to establish whether there is a link between early life Pb exposure and later THC-related behavior in C57BL6/J mice, as has been demonstrated for other drugs of abuse. The first aim seeks to answer whether Pb exposure affects physiological THC sensitivity (as measured by the cannabinoid-induced tetrad). The second aim seeks to answer whether Pb exposure affects edible THC self-administration and the development of THC tolerance (also measured by the tetrad). It was hypothesized that Pb exposure would decrease THC sensitivity (Aim 1), would enhance THC self-administration (Aim 2), enhance the development of THC tolerance (Aim 2), and finally that sex-dependent effects of Pb-exposure and THC would be observed (Aims 1 & 2). These hypotheses ended up not being supported, but Aim 1 produced findings indicating that THC sensitivity was increased by Pb exposure, but only in female mice. Future research will hopefully be able to fully explore the implications of these findings.

The etiology of many gestational disorders is still unknown. However, insufficient trans-placental passage of nutrients and wastes due to poor placentation is characteristic of several pathologies and may be due, in part, to altered function of placental mitochondria. Mitochondrial activity is essential in pregnancy because it sustains the metabolic activity of the placenta throughout gestation. Exposure to stressors that perturb processes governing placentation, including maternal drug use, can negatively impact fetal development. Cannabis use is prevalent during pregnancy. The psychoactive constituent, delta-9-tetrahydrocannbinol (THC), can cross the placenta to affect placental and fetal physiology. Importantly, cannabinoid receptors have been reported on trophoblast cells, and on mitochondria which are abundant in placentae. It has been reported that THC may target the mitochondria in various tissue types, including placental tissue, and alter its function. However, few studies have addressed the physiological control of mitochondria within the placenta, an organ that is critical for fetal growth and pregnancy maintenance. I investigated the role of mitochondria in trophoblast differentiation and syncytialization using rotenone, a complex I inhibitor. Subsequently, I investigated the role of THC on two important aspects of placentation – invasion and syncytialization – using placental trophoblast cells HTR8/SVneo and BeWo, respectively. In response to rotenone and THC, there was increased ROS production, oxidative stress, and altered transcriptional markers favouring mitochondrial fragmentation. Treatment with 20µM THC for 48 hours led to reduced mitochondrial respiration, ATP production and loss of mitochondrial membrane polarity. Critically, these THC-induced mitochondrial changes occurred concomitant with evidence of reduced trophoblast invasion and syncytialization. Furthermore, THC exposure reduced levels of human chorionic gonadotropin, human placental lactogen and insulin-like growth factor 2, which are growth factors necessary for fetal development. Placental mitochondrial dysfunction, particularly when THC-induced, may be critical in a range of gestational disorders which have important implications for maternal and fetal/offspring health. / Dissertation / Doctor of Philosophy (Medical Science) / Cannabis is commonly used by pregnant women. Fetal exposure to cannabis and its components can impair fetal growth and neurological development. These negative fetal outcomes may be the result of poor placental formation, due to placental cell exposure to cannabis and its psychoactive component, delta-9-tetrahydrocannabinol (THC). Importantly, THC can also target intracellular organelles, like the mitochondria which are known as the “powerhouses” of the cell. Few studies have investigated the direct effects of THC on placental development. The purpose of this study was to determine how THC exposure to placental cells may alter their function. We found that THC impaired processes that allow placental attachment to the uterus and form a protective barrier, and compromised mitochondrial function, which are important for placental formation. These findings serve to inform scientists and doctors, thus stimulating the creation of new ideas and methods to further explore the impact of THC on pregnancy outcomes.

Over the approximate nine months of its intrauterine existence, the development of the fetus is supported by the human placenta. This transient organ is central to pregnancy success as it facilitates maternal-fetal exchange, immunological tolerance, and hormone production. Villous trophoblasts mediate placental formation by engaging in a continuous turnover process of proliferation, differentiation, fusion, and apoptosis. In doing so, cytotrophoblasts and syncytiotrophoblasts maintain the integrity of the outer placental lining known as the syncytium. Exposure to drugs, however, can compromise placental establishment, which can in turn adversely impact pregnancy and fetal health. Specifically, cannabis is widely used by women of reproductive age and during pregnancy. While maternal cannabis use is linked to poor outcomes such as preterm birth and neurodevelopmental delays in exposed children, the underlying mechanisms are not well-defined. First, we characterized a functionally relevant cell line to model differentiation and fusion. In a comparison of the BeWo and BeWo b30 cell lines, our findings demonstrated that both models similarly undergo fusion. We then explored the implications of exposure to delta-9- tetrahydrocannabinol (∆9-THC) on the immunological roles of villous trophoblasts. We observed that cytotrophoblast differentiation and fusion were associated with localized inflammation due to elevated interleukin-2 (IL-2) and tumour necrosis factor-alpha (TNF-α) but inhibited interleukin-4 (IL-4) and interleukin-10 (IL-10) production. ∆9-THC exposure impaired this T helper 1/2 cytokine balance through decreased IL-2 and TNF-α as well as increased IL-4 and IL-10 levels. Subsequently, we investigated the effects of ∆9-THC in TNF-α- and IL-10-dominant environments, to represent inflammatory and immunomodulatory microenvironments, respectively. Coincident with inflammation, ∆9-THC attenuated trophoblast fusion and the biosynthesis of steroid hormones, progesterone and cortisol, through perturbed cytochrome P450 regulation. This thesis ultimately lays a foundation for understanding how cannabis use during pregnancy may compromise the fusogenic, immune and endocrine functions of villous trophoblasts in the placenta. / Thesis / Master of Science (MSc) / The human placenta is a pregnancy-specific organ that supports the health of the mother- to-be and fetus. Stem cells known as cytotrophoblasts undergo differentiation and fusion to support the establishment of the syncytium, which creates a boundary that separates the maternal and fetal circulations. In the case of cannabis consumption during pregnancy, its biologically active components can travel to the placenta, cross the syncytium, and enter fetal blood. Our primary objective was to determine how cannabis exposure can impact the formation and maintenance of the syncytium. While maternal use has been linked to short- and long-term consequences for child health, existing research lacks a complete understanding of the underlying mechanisms. We demonstrate that cannabis exposure alters the production of important immune and hormonal factors during cytotrophoblast fusion, which may play a role in mediating poor placental development. Ultimately, it is critical to explore the implications of cannabis use for female reproductive health due to a rising trend in its use.

<p>Environmental exposure to lead (Pb) and cannabis use are two of the largest public health issues facing modern society in the United States and around the world. Exposure to Pb in early life has been unequivocally shown to have negative impacts on development, and recent research is mounting showing that it may also predispose individuals for risk of developing substance use disorders (SUD). At the same time, societal and legal attitudes towards cannabis (main psychoactive component delta-9-tetrahydrocannabinol) have been shifting, and many American states have legalized the recreational use of cannabis. It is also the 3^rdmost widely used drug of abuse in the US, and rates of cannabis use disorder are on the rise. This thesis sets out to establish whether there is a link between early life Pbexposure and later THC-related behavior in C57BL6/J mice, as has been demonstrated for other drugs of abuse. The first aim seeks to answer whether Pbexposure affects physiological THC sensitivity (as measured by the cannabinoid-induced tetrad). The secondaimseeks to answer whether Pbexposure affects edible THC self-administration and the development of THC tolerance (also measured by the tetrad).It was hypothesized that Pbexposure would decrease THC sensitivity (Aim 1), would enhance THC self-administration (Aim 2), enhance the development of THC tolerance (Aim 2), and finally that sex-dependent effects of Pb-exposure and THC would be observed (Aims 1 & 2). These hypotheses ended up not being supported, but Aim 1 produced findings indicating that THC sensitivity was increased by Pbexposure, but only in female mice. Future researchwill hopefully be able to fully explore the implications of these findings.</p>

A maconha é a droga ilícita mais consumida no mundo, tem como substância psicoativa o delta-9-tetraidrocanabinol (9-THC). Os testes colorimétricos são normalmente realizados em amostras de maconha (e para qualquer outra droga ilícita) apreendidas. A adição de reagentes específicos em uma solução de extrato de maconha pode indicar a presença de substância ilícita mediante mudança de cor. No entanto, a literatura recentemente tem relatado ocorrências de resultados falso-positivos quando os testes colorimétricos são aplicados em algumas folhagens de diversas espécies de plantas. Este estudo utilizou as técnicas voltamétricas em meio orgânico N-N-dimetilformamida utilizando tetrafluoroborato de tetrabutilamônio como eletrólito de suporte e eletrodos de trabalho de disco de carbono vítreo e platina. Aplicando-se um potencial de pré concentração em - 0,5 V, verifica-se um pico de corrente anódica em ± 0,01 V vs Ag/AgCl ,KCl(sat). Utilizando eletrodo de disco de carbono vítreo como eletrodo de trabalho, na modalidade de onda quadrada, obteve-se uma dependência linear na faixa de concentração entre 1,0 x 10-9 mol L-1 a 2,2 x 10-8 mol L-1, com um coeficiente de correlação linear em 0,999 e um limite de detecção de 6,2 x 10-10 mol L-1. Tais resultados possibilitaram a determinação de 9-THC na ordem de nmol L-1. / Marijuana, the illicit drug that is most consumed worldwide, contains 9-tetrahydrocannabinol (9-THC) as the main psychoactive substance. Presumptive colorimetric tests are usually performed on seized marijuana (or any illicit drug) samples: the addition of specific reagents to the marijuana extract solution prompts a change in. This study was developed using voltammetric techniques in organic medium (N,N-dimethylformamide) using tetrabutylammonium tetrafluoroborate (TBATFB) as supporting electrolyte, and glassy carbon and platinum disc as working electrode. Applying a pre-concentration potential of -0.5 by square-wave voltammetry, we detected a well-defined anodic peak current in ± 0.01 V versus Ag/AgCl, KCl(sat); 9-THC detection presented linear dependence at concentrations ranging from 1.0 × 10-9 mol L-1 to 2.2 × 10-8 mol L-1, with a linear correlation coefficient 0.999 and a detection limit of 6.2 × 10-10 mol L-1, using the glassy carbon disc working electrode.

A maconha é a droga ilícita mais consumida no mundo, tem como substância psicoativa o delta-9-tetraidrocanabinol (9-THC). Os testes colorimétricos são normalmente realizados em amostras de maconha (e para qualquer outra droga ilícita) apreendidas. A adição de reagentes específicos em uma solução de extrato de maconha pode indicar a presença de substância ilícita mediante mudança de cor. No entanto, a literatura recentemente tem relatado ocorrências de resultados falso-positivos quando os testes colorimétricos são aplicados em algumas folhagens de diversas espécies de plantas. Este estudo utilizou as técnicas voltamétricas em meio orgânico N-N-dimetilformamida utilizando tetrafluoroborato de tetrabutilamônio como eletrólito de suporte e eletrodos de trabalho de disco de carbono vítreo e platina. Aplicando-se um potencial de pré concentração em - 0,5 V, verifica-se um pico de corrente anódica em ± 0,01 V vs Ag/AgCl ,KCl(sat). Utilizando eletrodo de disco de carbono vítreo como eletrodo de trabalho, na modalidade de onda quadrada, obteve-se uma dependência linear na faixa de concentração entre 1,0 x 10-9 mol L-1 a 2,2 x 10-8 mol L-1, com um coeficiente de correlação linear em 0,999 e um limite de detecção de 6,2 x 10-10 mol L-1. Tais resultados possibilitaram a determinação de 9-THC na ordem de nmol L-1. / Marijuana, the illicit drug that is most consumed worldwide, contains 9-tetrahydrocannabinol (9-THC) as the main psychoactive substance. Presumptive colorimetric tests are usually performed on seized marijuana (or any illicit drug) samples: the addition of specific reagents to the marijuana extract solution prompts a change in. This study was developed using voltammetric techniques in organic medium (N,N-dimethylformamide) using tetrabutylammonium tetrafluoroborate (TBATFB) as supporting electrolyte, and glassy carbon and platinum disc as working electrode. Applying a pre-concentration potential of -0.5 by square-wave voltammetry, we detected a well-defined anodic peak current in ± 0.01 V versus Ag/AgCl, KCl(sat); 9-THC detection presented linear dependence at concentrations ranging from 1.0 × 10-9 mol L-1 to 2.2 × 10-8 mol L-1, with a linear correlation coefficient 0.999 and a detection limit of 6.2 × 10-10 mol L-1, using the glassy carbon disc working electrode.

O Canabidiol (CBD), que representa aproximadamente 40% dos canabinóides encontrados na planta Cannabis sativa, é desprovido dos efeitos psicológicos e cognitivos típicos do 9-Tetraidrocanabinol (9THC). Estudos sugerem que o CBD apresenta propriedades ansiolíticas, porém esta substância nunca foi testada na ansiedade clínica. Do mesmo modo, não se sabe como estes possíveis efeitos seriam mediados centralmente em pacientes com transtorno de ansiedade social (TAS). Diante das evidências da existência de um sistema canabinóide em humanos e do crescente interesse terapêutico no uso do CBD, justifica-se um estudo para desenvolvimento e validação de uma metodologia analítica para a determinação e quantificação de CBD e 9THC empregando a técnica de cromatografia em fase gasosa/espectrometria de massas. O método desenvolvido e validado se mostrou rápido, simples, de baixo custo com boa sensibilidade e apropriado para aplicação na área da toxicologia clínica. O método demonstrou ser linear no intervalo de concentração de 5 a 500 ng/0,5 mL de plasma para o CBD (r2 = 0,99) e de 5 a 300 ng/0,5 mL (r2 = 0,98) para o 9THC. Os limites de detecção e quantificação foram respectivamente 0,1 ng/0,5 mL e 0,5 ng/0,5 mL para o CBD e, 5 ng/0,5 mL e 10 ng/0,5 mL para o 9THC. Valores de precisão inter e intra ensaio estão respectivamente, na faixa de 5,5% a 12,7%, e de 2,1% a 8,1%. Valores de exatidão inter e intra ensaio estão respectivamente, na faixa de 1,2% a 12,0, e de 1,2 a 14,5 para CBD e 9THC. A eficiência da extração foi obtida na faixa de 54,6 a 93,2% de recuperação para os analitos. A metodologia validada foi empregada em um estudo clínico para correlacionar a dose após a administração controlada de CBD em pacientes com transtorno de ansiedade social. / Dissertation (Master). Faculdade de Ciências Farmacêuticas de Ribeirão Preto Universidade de São Paulo, Ribeirão Preto, 2008. The Cannabidiol (CBD), that represents about 40% of the cannabinoids found in Cannabis sativa plant, is devoided of the typical psychological and cognitive effects of 9-Tetrahydrocannabinol (9THC). Researches suggest that CBD shows ansiolitic properties, but this substance was never tested on clinical anxiety. It is unknown how possible CBD effects could be centrally mediated in social anxiety disorder (SAD) patients. There are evidences about the existence of a cannabinoid system in humans and there is also increasing interest on therapeutic CBD application. The present study was conducted to develop and validate an analytical methodology for determination and quantification of CBD and 9THC, employing gas phase chromatography/mass spectrometry technique. The validated method was fast, easy, low cost, with a good sensibility and appropriate for clinical toxicology applications. The method was linear on the range of 5 to 500 ng/0,5 mL of plasma for CBD (r2=0,99) and 5 to 300 ng/0,5 mL (r2=0,98) for 9THC. The detection and quantification limits were 0,1 ng/0,5 mL and 0,5 ng/0,5 mL for CBD and 5ng/0,5 mL and 10 ng/0,5 mL for 9THC respectively. Inter and intraday reproducibility were between 5,5% to 12,7% and 2,1% to 8,1%, respectively. Inter and intraday accuracy was between 1,2% to 12% and 1,2% to 14,5% for CBD and 9THC respectively. The recovery of extraction was between 54, 6% to 93,2% of recovery for the analytes The validated methodology was applied in a clinical trial to correlate the doses after the controlled cannabidiol administration to patients with social anxiety disorders.

O Canabidiol (CBD), que representa aproximadamente 40% dos canabinóides encontrados na planta Cannabis sativa, é desprovido dos efeitos psicológicos e cognitivos típicos do 9-Tetraidrocanabinol (9THC). Estudos sugerem que o CBD apresenta propriedades ansiolíticas, porém esta substância nunca foi testada na ansiedade clínica. Do mesmo modo, não se sabe como estes possíveis efeitos seriam mediados centralmente em pacientes com transtorno de ansiedade social (TAS). Diante das evidências da existência de um sistema canabinóide em humanos e do crescente interesse terapêutico no uso do CBD, justifica-se um estudo para desenvolvimento e validação de uma metodologia analítica para a determinação e quantificação de CBD e 9THC empregando a técnica de cromatografia em fase gasosa/espectrometria de massas. O método desenvolvido e validado se mostrou rápido, simples, de baixo custo com boa sensibilidade e apropriado para aplicação na área da toxicologia clínica. O método demonstrou ser linear no intervalo de concentração de 5 a 500 ng/0,5 mL de plasma para o CBD (r2 = 0,99) e de 5 a 300 ng/0,5 mL (r2 = 0,98) para o 9THC. Os limites de detecção e quantificação foram respectivamente 0,1 ng/0,5 mL e 0,5 ng/0,5 mL para o CBD e, 5 ng/0,5 mL e 10 ng/0,5 mL para o 9THC. Valores de precisão inter e intra ensaio estão respectivamente, na faixa de 5,5% a 12,7%, e de 2,1% a 8,1%. Valores de exatidão inter e intra ensaio estão respectivamente, na faixa de 1,2% a 12,0, e de 1,2 a 14,5 para CBD e 9THC. A eficiência da extração foi obtida na faixa de 54,6 a 93,2% de recuperação para os analitos. A metodologia validada foi empregada em um estudo clínico para correlacionar a dose após a administração controlada de CBD em pacientes com transtorno de ansiedade social. / Dissertation (Master). Faculdade de Ciências Farmacêuticas de Ribeirão Preto Universidade de São Paulo, Ribeirão Preto, 2008. The Cannabidiol (CBD), that represents about 40% of the cannabinoids found in Cannabis sativa plant, is devoided of the typical psychological and cognitive effects of 9-Tetrahydrocannabinol (9THC). Researches suggest that CBD shows ansiolitic properties, but this substance was never tested on clinical anxiety. It is unknown how possible CBD effects could be centrally mediated in social anxiety disorder (SAD) patients. There are evidences about the existence of a cannabinoid system in humans and there is also increasing interest on therapeutic CBD application. The present study was conducted to develop and validate an analytical methodology for determination and quantification of CBD and 9THC, employing gas phase chromatography/mass spectrometry technique. The validated method was fast, easy, low cost, with a good sensibility and appropriate for clinical toxicology applications. The method was linear on the range of 5 to 500 ng/0,5 mL of plasma for CBD (r2=0,99) and 5 to 300 ng/0,5 mL (r2=0,98) for 9THC. The detection and quantification limits were 0,1 ng/0,5 mL and 0,5 ng/0,5 mL for CBD and 5ng/0,5 mL and 10 ng/0,5 mL for 9THC respectively. Inter and intraday reproducibility were between 5,5% to 12,7% and 2,1% to 8,1%, respectively. Inter and intraday accuracy was between 1,2% to 12% and 1,2% to 14,5% for CBD and 9THC respectively. The recovery of extraction was between 54, 6% to 93,2% of recovery for the analytes The validated methodology was applied in a clinical trial to correlate the doses after the controlled cannabidiol administration to patients with social anxiety disorders.

This thesis describes in the form of a commentary on own original publications research on the problems of cannabinoids, ie. phytocannabinoids and some synthetic cannabinoids, their pharmacokinetics and effects. The work consists of four thematic areas: the pharmacokinetics of delta-9- tetrahydrocannabinol (THC) and cannabidiol (CBD) in rats, depending on the route of administration; THC concentration time profile in humans (after inhalation) and implications for transport safety; the pharmacokinetic profile of synthetic cannabinoids in rats; extraction and determination of phytocannabinoids in plant material. The first part of the thesis was to determine pharmacokinetic profiles of THC, CBD and combination thereof (1:1 weight ratio) in rats with respect to administration common in humans, i.e. inhalation, oral and subcutaneous administration. THC, its metabolites (11-hydroxy-tetrahydrocannabinol, 11-OH-THC; 11-nor-delta-9- carboxytetrahydrocannabinol, THCOOH) and CBD concentrations in serum and brains of animals were monitored at the 24 hours experimental interval during the study. Except for inhalation administration, co-administration of CBD inhibited THC metabolism (after both oral and subcutaneous), resulting in an increase in THC concentrations in both serum and brain of the rats relative to...

The principal psychoactive component of marihuana is Δ-9-tetrahy-drocannabinol. This compound at 10-5 molar concentration in the medium of human cell cultures appeared to inhibit DNA, RNA, and protein synthesis by 50, 40, and 30% respectively, as measured by incorporation of radioactive precursors into acid-insoluble cell fractions in human diploid fibroblasts, human neuroblastoma cells, and mouse neuroblastoma cells. While Δ-9-tetrahydrocannabinol inhibited semiconservative DNA synthesis, it had no effect on DNA repair synthesis in human cells as assayed by the photolysis of 5-bromodeoxyuridine incorporation into DNA during repair after ultraviolet radiation damage. Δ-9-tetrahydrocannabinol also had no effect on rejoining of DNA single-strand breaks induced by γ-rays. The nonspecificity of the inhibition of macromolecular synthesis by Δ-9-THC suggested a possible interference with uptake of radioactive precursors. However, experimentation has shown that this depression of macromolecular synthesis cannot be accounted for by reduced transport of radioactive precursors into the cell because the rate of transport of these precursors into the cell is essentially the same in the presence or absence of Δ-9-THC. Pool sizes of macromolecular precursors as measured radioisotopically (3Hthymidine, 3H-uridine, 14C-leucine) appear to be reduced about 50%, and this reduced pool size could fully account for the reduced macromolecular synthesis seen in the presence of Δ-9-THC. We do not know what causes this apparent reduction of pool sizes in the presence of Δ-9-THC.