Oral exposure to polyethylene microplastics induces inflammatory and metabolic changes and promotes fibrosis in mouse liver.

Accumulating evidence shows widespread contamination of water sources and food with microplastics. Although the liver is one of the main sites of bioaccumulation within the human body, it is still unclear whether microplastics produce damaging effects. In particular, the hepatic consequences of ingesting polyethylene (PE) microplastics in mammals are unknown. In this study, female mice were fed with food contaminated with 36 and 116 µm diameter PE microbeads at a dosage of 100 µg/g of food for 6 and 9 weeks. Mice were exposed to each type of microbead, or co-exposed to the 2 types of microbeads. Mouse liver showed altered levels of genes involved in uptake, synthesis, and ß-oxidation of fatty acids. Ingestion of PE microbeads disturbed the detoxification response, promoted oxidative imbalance, increased inflammatory foci and cytokine expression, and enhanced proliferation in liver. Since relative expression of the hepatic stellate cell marker Pdgfa and collagen deposition were increased following PE exposure, we assessed the effect of PE ingestion in a mouse model of CCl4-induced fibrosis and showed that PE dietary exposure exacerbated liver fibrogenesis. These findings provide the first demonstration of the adverse hepatic effects of PE ingestion in mammals and highlight the need for further health risk assessment in humans.

Oral exposure to polyethylene microplastics alters gut morphology, immune response, and microbiota composition in mice.

The ubiquitous and growing presence of microplastics (MPs) in all compartments of the environment raises concerns about their possible harmful effects on human health. Human exposure to MPs occurs largely through ingestion. Polyethylene (PE) is widely employed for reusable bags and food packaging and found to be present in drinking water and food. It is also one of the major polymers detected in human stool. The aim of this study was to characterize the effects of intestinal exposure to PE MPs on gut homeostasis. Mice were orally exposed for 6 weeks to PE microbeads of 2 different sizes, 36 and 116 µm, that correspond to those found in human stool. They were administrated either individually or as a mixture at a dose of 100 µg/g of food. Both PE microbead sizes were detected in mouse stool. Different parameters related to major intestinal functions were compared between control mice, mice exposed to each type of microbead, or co-exposed to the 2 types of microbeads. Intestinal disturbances were observed after individual exposure to each size of PE microbead, and the most marked deleterious effects were found in co-exposed mice. At the histomorphological level, crypt depth was increased throughout the intestinal tissues. Significant variations of gene expression related to epithelial, permeability, and inflammatory biomarkers were quantified. Defective recruitment of some intestinal immune cells was observed from the proximal portion of the small intestine to the colon. Several bacterial taxa at the order level were found to be affected by exposure to the MPs by metagenomic analysis of cecal microbiota. These results show that ingestion of PE microbeads induces significant alterations of crucial intestinal markers in mice and underscores the need to further study the health impact of MP exposure in humans.

Murine in utero exposure to simulated complex urban air pollution disturbs offspring gut maturation and microbiota during intestinal suckling-to-weaning transition in a sex-dependent manner.

BACKGROUND: Emerging data indicate that prenatal exposure to air pollution may lead to higher susceptibility to several non-communicable diseases. Limited research has been conducted due to difficulties in modelling realistic air pollution exposure. In this study, pregnant mice were exposed from gestational day 10-17 to an atmosphere representative of a 2017 pollution event in Beijing, China. Intestinal homeostasis and microbiota were assessed in both male and female offspring during the suckling-to-weaning transition. RESULTS: Sex-specific differences were observed in progeny of gestationally-exposed mice. In utero exposed males exhibited decreased villus and crypt length, vacuolation abnormalities, and lower levels of tight junction protein ZO-1 in ileum. They showed an upregulation of absorptive cell markers and a downregulation of neonatal markers in colon. Cecum of in utero exposed male mice also presented a deeply unbalanced inflammatory pattern. By contrast, in utero exposed female mice displayed less severe intestinal alterations, but included dysregulated expression of Lgr5 in colon, Tjp1 in cecum, and Epcam, Car2 and Sis in ileum. Moreover, exposed female mice showed dysbiosis characterized by a decreased weighted UniFrac ß-diversity index, a higher abundance of Bacteroidales and Coriobacteriales orders, and a reduced Firmicutes/Bacteroidetes ratio. CONCLUSION: Prenatal realistic modelling of an urban air pollution event induced sex-specific precocious alterations of structural and immune intestinal development in mice.

Exposure to atmospheric Ag, TiO2, Ti and SiO2 engineered nanoparticles modulates gut inflammatory response and microbiota in mice.

The development of nanotechnologies is leading to greater abundance of engineered nanoparticles (EN) in the environment, including in the atmospheric air. To date, it has been shown that the most prevalent EN found in the air are silver (Ag), titanium dioxide (TiO2), titanium (Ti), and silicon dioxide (SiO2). As the intestinal tract is increasingly recognized as a target for adverse effects induced by inhalation of air particles, the aim of this study was to assess the impact of these 4 atmospheric EN on intestinal inflammation and microbiota. We assessed the combined toxicity effects of Ag, Ti, TiO2, and SiO2 following a 28-day inhalation protocol in male and female mice. In distal and proximal colon, and in jejunum, EN mixture inhalation did not induce overt histological damage, but led to a significant modulation of inflammatory cytokine transcript abundance, including downregulation of Tnfα, Ifnγ, Il1ß, Il17a, Il22, IL10, and Cxcl1 mRNA levels in male jejunum. A dysbiosis was observed in cecal microbiota of male and female mice exposed to the EN mixture, characterized by sex-dependent modulations of specific bacterial taxa, as well as sex-independent decreased abundance of the Eggerthellaceae family. Under dextran sodium sulfate-induced inflammatory conditions, exposure to the EN mixture increased the development of colitis in both male and female mice. Moreover, the direct dose-response effects of individual and mixed EN on gut organoids was studied and Ag, TiO2, Ti, SiO2, and EN mixture were found to generate specific inflammatory responses in the intestinal epithelium. These results indicate that the 4 most prevalent atmospheric EN could have the ability to disturb intestinal homeostasis through direct modulation of cytokine expression in gut epithelium, and by altering the inflammatory response and microbiota composition following inhalation.

Chronic ingestion of deoxynivalenol at human dietary levels impairs intestinal homeostasis and gut microbiota in mice.

The mycotoxin deoxynivalenol (DON) is a frequent contaminant of cereals and their by-products in areas with a moderate climate. Produced by Fusarium species, it is one of the most prevalent mycotoxins in cereal crops worldwide, and the most frequently occurring type B trichothecene in Europe. Due to its toxic properties, high stability and prevalence, the presence of DON in the food chain could represent a major public health risk. However, despite its well-known acute toxicological effects, information on the adverse effects of realistic exposure remains limited. We orally exposed mice during 9 months to DON at doses relevant for currently estimated human intake and explored the impact on various gut health parameters. DON exposure induced recruitment of regulatory B cells, and activation of regulatory T cells and dendritic cells in mesenteric lymph nodes. Several inflammatory parameters were increased in colon of DON-exposed mice, whereas inversely inflammatory markers were decreased in ileum. Histomorphological impairments were observed from the duodenum to the colon. Both colon and jejunum presented a hyperproliferation of epithelial cells and an increased expression of mature absorptive cells markers. Finally, DON exposure reshaped gut microbial structure and drastically disturbed the abundance of several bacterial phyla, families, and genera, leading to dysbiosis. Chronic oral exposure to human relevant doses of DON induces several disturbances of gut homeostasis with likely pathological implications for susceptible individuals.

Effects of urban coarse particles inhalation on oxidative and inflammatory parameters in the mouse lung and colon.

BACKGROUND: Air pollution is a recognized aggravating factor for pulmonary diseases and has notably deleterious effects on asthma, bronchitis and pneumonia. Recent studies suggest that air pollution may also cause adverse effects in the gastrointestinal tract. Accumulating experimental evidence shows that immune responses in the pulmonary and intestinal mucosae are closely interrelated, and that gut-lung crosstalk controls pathophysiological processes such as responses to cigarette smoke and influenza virus infection. Our first aim was to collect urban coarse particulate matter (PM) and to characterize them for elemental content, gastric bioaccessibility, and oxidative potential; our second aim was to determine the short-term effects of urban coarse PM inhalation on pulmonary and colonic mucosae in mice, and to test the hypothesis that the well-known antioxidant N-acetyl-L-cysteine (NAC) reverses the effects of PM inhalation. RESULTS: The collected PM had classical features of urban particles and possessed oxidative potential partly attributable to their metal fraction. Bioaccessibility study confirmed the high solubility of some metals at the gastric level. Male mice were exposed to urban coarse PM in a ventilated inhalation chamber for 15 days at a concentration relevant to episodic elevation peak of air pollution. Coarse PM inhalation induced systemic oxidative stress, recruited immune cells to the lung, and increased cytokine levels in the lung and colon. Concomitant oral administration of NAC reversed all the observed effects relative to the inhalation of coarse PM. CONCLUSIONS: Coarse PM-induced low-grade inflammation in the lung and colon is mediated by oxidative stress and deserves more investigation as potentiating factor for inflammatory diseases.

Intestinal steroidogenesis controls PPARγ expression in the colon and is impaired during ulcerative colitis.

BACKGROUND AND AIMS: Immune tolerance breakdown during UC involves the peroxisome proliferator-activated receptor-γ (PPARγ), a key factor in mucosal homoeostasis and the therapeutic target of 5-aminosalycilates, which expression is impaired during UC. Here we assess the impact of glucocorticoids (GCs) on PPARγ expression, focusing especially on extra-adrenal cortisol production by colonic epithelial cells (CECs). METHODS: Activation of PPARγ in the colon was evaluated using transgenic mice for the luciferase gene under PPAR control (peroxisome proliferator response element-luciferase mice). Protein and mRNA expression of PPARγ were evaluated with colon fragments and purified CEC from mice. Cortisol production and steroidogenic factor expression were quantified in human CEC of patients with UC and those of controls. Gene expression knockdown by short hairpin RNA in Caco-2 cells was used for functional studies. RESULTS: GCs were able to raise luciferase activity in peroxisome proliferator response element-luciferase mice. In the mice colons and Caco-2 cells, PPARγ expression was increased either with GCs or with an inducer of steroidogenesis and then decreased after treatment with a steroidogenesis inhibitor. Cortisol production and steroidogenic factor expression, such as liver receptor homologue-1 (LRH-1), were decreased in CEC isolated from patients with UC, directly correlating with PPARγ impairment. Experiments on Caco-2 cells lacking LRH-1 expression confirmed that LRH-1 controls PPARγ expression by regulating GC synthesis in CEC. CONCLUSIONS: These results demonstrate cortisol control of PPARγ expression in CEC, highlighting cortisol production deficiency in colonocytes as a key molecular event in the pathophysiology of UC.

Switching cannabinoid response from CB(2) agonists to FAAH inhibitors.

A series of 3-carboxamido-5-aryl-isoxazoles designed as CB2 agonists were evaluated as FAAH inhibitors. The pharmacological results led to identify structure-activity relationships enabling to switch cannabinoid response from CB2 agonists to FAAH inhibitors. Two compounds were selected for their FAAH and/or CB2 activity, and evaluated in a colitis model for their anti-inflammatory activity. Results showed that compounds 10 and 11 inhibit the development of DSS-induced acute colitis in mice and then, are interesting leads to explore new drug candidates for IBD.

3-Carboxamido-5-aryl-isoxazoles as new CB2 agonists for the treatment of colitis.

Recent investigations showed that anandamide, the main endogenous ligand of CB1 and CB2 cannabinoid receptors, possesses analgesic, antidepressant and anti-inflammatory effects. In the perspective to treat inflammatory bowel disease (IBD), our approach was to develop new selective CB2 receptor agonists without psychotropic side effects associated to CB1 receptors. In this purpose, a new series of 3-carboxamido-5-aryl-isoxazoles, never described previously as CB2 receptor agonists, was designed, synthesized and evaluated for their biological activity. The pharmacological results have identified great selective CB2 agonists with in vivo anti-inflammatory activity in a DSS-induced acute colitis mouse model.

Low dose dietary contamination with deoxynivalenol mycotoxin exacerbates enteritis and colorectal cancer in mice.

BACKGROUND: The mycotoxin deoxynivalenol (DON) is a frequent contaminant of grain and cereal products worldwide. Exposure to DON can cause gastrointestinal inflammation, disturb gut barrier function, and induce gut dysbiosis in vivo under basal conditions, but little is known about the effects of DON ingestion in individuals with pre-existing gastrointestinal disease. OBJECTIVES: Mice were orally exposed to 10 and 100 µg/kg bw/day of DON, corresponding to 10 to 100-fold human tolerable daily intake concentrations, and to the translation in mice of current human daily intake. The effects of DON exposure were explored under steady-state conditions, and in murine models of enteritis and colorectal cancer (CRC). RESULTS: After 8 days of DON exposure, an increase of histomorphological and molecular parameters of epithelial proliferation were observed in normal mice, from the duodenum to the colon. The same exposure in a murine model of indomethacin-induced enteritis led to exacerbation of lesion development and induction of ileal cytokines. DON exposure also worsened the development of colitis-associated CRC in mice as shown by increases in endoscopic and histological colitis scores, tumor grades, and histological hyperplasia. In colon of DON-exposed mice, upstream and downstream ERK signaling genes were upregulated including Mapk1, Mapk3, Map 2k1, Map2k2 core ERK pathway effectors, and Bcl2 and Bcl2l1 antiapoptotic genes. The effects observed in the CRC model were associated with alterations in cecal microbiota taxonomic composition and metabolism of bacterial fucose and rhamnose. Strong Spearman's correlations were revealed between the relative abundance of the changed bacterial genera and CRC-related variables. DISCUSSION: Ingestion of DON mycotoxin at concentrations representative of human real-world exposure worsened the development of indomethacin-induced enteritis and colitis-associated CRC in mice. Our results suggest that even at low doses, which are currently tolerated in the human diet, DON could promote the development of intestinal inflammatory diseases and CRC.

Thymidylate synthase O-GlcNAcylation: a molecular mechanism of 5-FU sensitization in colorectal cancer.

Alteration of O-GlcNAcylation, a dynamic posttranslational modification, is associated with tumorigenesis and tumor progression. Its role in chemotherapy response is poorly investigated. Standard treatment for colorectal cancer (CRC), 5-fluorouracil (5-FU), mainly targets Thymidylate Synthase (TS). TS O-GlcNAcylation was reported but not investigated yet. We hypothesize that O-GlcNAcylation interferes with 5-FU CRC sensitivity by regulating TS. In vivo, we observed that combined 5-FU with Thiamet-G (O-GlcNAcase (OGA) inhibitor) treatment had a synergistic inhibitory effect on grade and tumor progression. 5-FU decreased O-GlcNAcylation and, reciprocally, elevation of O-GlcNAcylation was associated with TS increase. In vitro in non-cancerous and cancerous colon cells, we showed that 5-FU impacts O-GlcNAcylation by decreasing O-GlcNAc Transferase (OGT) expression both at mRNA and protein levels. Reciprocally, OGT knockdown decreased 5-FU-induced cancer cell apoptosis by reducing TS protein level and activity. Mass spectrometry, mutagenesis and structural studies mapped O-GlcNAcylated sites on T251 and T306 residues and deciphered their role in TS proteasomal degradation. We reveal a crosstalk between O-GlcNAcylation and 5-FU metabolism in vitro and in vivo that converges to 5-FU CRC sensitization by stabilizing TS. Overall, our data propose that combining 5-FU-based chemotherapy with Thiamet-G could be a new way to enhance CRC response to 5-FU.

Gene/environment interaction in the susceptibility of Crohn's disease patients to aluminum.

BACKGROUND & AIM: The key role of environmental factors in the pathogenesis of Inflammatory Bowel Diseases (IBD) is recognized. Aluminum is suspected to be a risk factor for IBD. However, mechanisms linking aluminum exposure to disease development are unknown. We examined the role of aluminum transport and subcellular localisation on human colon susceptibility to aluminum-induced inflammation. METHODS: Human colon biopsies isolated from Crohn's disease (CD) or control patients and Caco-2 cells were incubated with aluminum. The effects of aluminum were evaluated on cytokine secretion and transporter expression. The role of aluminum kinetics parameters was studied in Caco-2 using transport inhibitors and in human colon biopsies by assessing genetic polymorphisms of transporters. RESULTS: Aluminum exposure was shown to induce cytokine secretion in colon of CD but not healthy patients. In Caco-2 cells, aluminum internalisation was correlated with inflammatory status. In human colon, analysis of genetic polymorphisms and expression of ABCB1 and SLC26A3 transporters showed that their decreased activity was involved in aluminum-induced inflammation. CONCLUSIONS: We hypothesize that alteration in detoxifying response would lead to a deregulation of intestinal homeostasis and to the expression of IBD. Our study emphasizes the complexity of gene/environment interaction for aluminum adverse health effect, highlighting at risk populations or subtypes of patients. A better understanding of correlations between gene expression or SNP and xenobiotic kinetics parameters would shift the medical paradigm to more personalized disease management and treatment.

New FAAH inhibitors based on 3-carboxamido-5-aryl-isoxazole scaffold that protect against experimental colitis.

Growing evidence suggests a role for the endocannabinoid (EC) system, in intestinal inflammation and compounds inhibiting anandamide degradation offer a promising therapeutic option for the treatment of inflammatory bowel diseases. In this paper, we report the first series of carboxamides derivatives possessing FAAH inhibitory activities. Among them, compound 39 displayed significant inhibitory FAAH activity (IC(50)=0.088 µM) and reduced colitis induced by intrarectal administration of TNBS.

O-GlcNAcylation Links Nutrition to the Epigenetic Downregulation of UNC5A during Colon Carcinogenesis.

While it is now accepted that nutrition can influence the epigenetic modifications occurring in colorectal cancer (CRC), the underlying mechanisms are not fully understood. Among the tumor suppressor genes frequently epigenetically downregulated in CRC, the four related genes of the UNC5 family: UNC5A, UNC5B, UNC5C and UNC5D encode dependence receptors that regulate the apoptosis/survival balance. Herein, in a mouse model of CRC, we found that the expression of UNC5A, UNC5B and UNC5C was diminished in tumors but only in mice subjected to a High Carbohydrate Diet (HCD) thus linking nutrition to their repression in CRC. O-GlcNAcylation is a nutritional sensor which has enhanced levels in CRC and regulates many cellular processes amongst epigenetics. We then investigated the putative involvement of O-GlcNAcylation in the epigenetic downregulation of the UNC5 family members. By a combination of pharmacological inhibition and RNA interference approaches coupled to RT-qPCR (Reverse Transcription-quantitative Polymerase Chain Reaction) analyses, promoter luciferase assay and CUT&RUN (Cleavage Under Target & Release Using Nuclease) experiments, we demonstrated that the O-GlcNAcylated form of the histone methyl transferase EZH2 (Enhancer of Zeste Homolog 2) represses the transcription of UNC5A in human colon cancer cells. Collectively, our data support the hypothesis that O-GlcNAcylation could represent one link between nutrition and epigenetic downregulation of key tumor suppressor genes governing colon carcinogenesis including UNC5A.

Aluminum Ingestion Promotes Colorectal Hypersensitivity in Rodents.

Background & Aims: Irritable bowel syndrome (IBS) is a multifactorial disease arising from a complex interplay between genetic predisposition and environmental influences. To date, environmental triggers are not well known. Aluminum is commonly present in food, notably by its use as food additive. We investigated the effects of aluminum ingestion in rodent models of visceral hypersensitivity, and the mechanisms involved. Methods: Visceral hypersensitivity was recorded by colorectal distension in rats administered with oral low doses of aluminum. Inflammation was analyzed in the colon of aluminum-treated rats by quantitative PCR for cytokine expression and by immunohistochemistry for immune cells quantification. Involvement of mast cells in the aluminum-induced hypersensitivity was determined by cromoglycate administration of rats and in mast cell-deficient mice (KitW-sh/W-sh). Proteinase-activated receptor-2 (PAR2) activation in response to aluminum was evaluated and its implication in aluminum-induced hypersensitivity was assessed in PAR2 knockout mice. Results: Orally administered low-dose aluminum induced visceral hypersensitivity in rats and mice. Visceral pain induced by aluminum persisted over time even after cessation of treatment, reappeared and was amplified when treatment resumed. As observed in humans, female animals were more sensitive than males. Major mediators of nociception were up-regulated in the colon by aluminum. Activation of mast cells and PAR2 were required for aluminum-induced hypersensitivity. Conclusions: These findings indicate that oral exposure to aluminum at human dietary level reproduces clinical and molecular features of IBS, highlighting a new pathway of prevention and treatment of visceral pain in some susceptible patients.

Benzo[d]thiazol-2(3H)-ones as new potent selective CB2 agonists with anti-inflammatory properties.

The high distribution of CB2 receptors in immune cells suggests their important role in the control of inflammation. Growing evidence offers this receptor as an attractive therapeutic target: selective CB2 agonists are able to modulate inflammation without triggering psychotropic effects. In this work, we report a new series of selective CB2 agonists based on a benzo[d]thiazol-2(3H)-one scaffold. This drug design project led to the discovery of compound 9, as a very potent CB2 agonist (Ki = 13.5 nM) with a good selectivity versus CB1. This compound showed no cytotoxicity, acceptable ADME-Tox parameters and demonstrates the ability to counteract colon inflammatory process in vivo.

Conformational Restriction Leading to a Selective CB2 Cannabinoid Receptor Agonist Orally Active Against Colitis.

The CB2 cannabinoid receptor has been implicated in the regulation of intestinal inflammation. Following on from the promising activity of a series of 4-oxo-1,4-dihydroquinoline-3-carboxamide, we developed constrained analogues based on a 2H-pyrazolo[4,3-c]quinolin-3(5H)-one scaffold, with improved affinity for the hCB2 receptor and had very high selectivity over the hCB1 receptor. Importantly, the lead of this series (26, hCB2: K i = 0.39 nM, hCB1: K i > 3000 nM) was found to protect mice against experimental colitis after oral administration.

AIEC colonization and pathogenicity: influence of previous antibiotic treatment and preexisting inflammation.

BACKGROUND: Inflammatory bowel diseases (IBD) patients are abnormally colonized by adherent-invasive Escherichia coli (AIEC). NOD2 gene mutations impair intracellular bacterial clearance. We evaluated the impact of antibiotic treatment on AIEC colonization in wildtype (WT) and NOD2 knockout mice (NOD2KO) and the consequences on intestinal inflammation. METHODS: After 3 days of antibiotic treatment, mice were infected for 2 days with 109 CFU AIEC and sacrificed 1, 5, and 60 days later. In parallel, mice were challenged with AIEC subsequent to a dextran sodium sulfate (DSS) treatment and sacrificed 9 days later. Ileum, colon, and mesenteric tissues were sampled for AIEC quantification and evaluation of inflammation. RESULTS: Without antibiotic treatment, AIEC was not able to colonize WT and NOD2KO mice. Compared with nontreated animals, antibiotic treatment led to a significant increase in ileal and colonic colonization of AIEC in WT and/or NOD2KO mice. Persistent AIEC colonization was observed until day 5 only in NOD2KO mice, disappearing at day 60. Mesenteric translocation of AIEC was observed only in NOD2KO mice. No inflammation was observed in WT and NOD2KO mice treated with antibiotics and infected with AIEC. During DSS-induced colitis, colonization and persistence of AIEC was observed in the colon. Moreover, a dramatic increase in clinical, histological, and molecular parameters of colitis was observed in mice infected with AIEC but not with a commensal E. coli strain. CONCLUSIONS: Antibiotic treatment was necessary for AIEC colonization of the gut and mesenteric tissues and persistence of AIEC was dependent on NOD2. AIEC exacerbated a preexisting DSS-induced colitis in WT mice.

4-Oxo-1,4-dihydropyridines as selective CB2 cannabinoid receptor ligands. Part 2: discovery of new agonists endowed with protective effect against experimental colitis.

Further on to our earlier work on the 4-oxo-1,4-dihydropyridine, we describe herein our strategy to get access to potent selective CB2 receptor agonists. Thus, we designed and synthesized 29 compounds, evaluated on both hCB1 and hCB2 cannabinoid receptors, and assessed 11 of them in the TNBS-induced colitis model in mice. Compound 48 was found to be the most efficient of our series, exhibiting an exquisite protection against experimental colitis, superior to the one observed after treatment with Pentasa.

4-Oxo-1,4-dihydropyridines as selective CB2 cannabinoid receptor ligands: structural insights into the design of a novel inverse agonist series.

Growing evidence shows that CB(2) receptor is an attractive therapeutic target. Starting from a series of 4-oxo-1,4-dihydroquinoline-3-carboxamide as selective CB(2) agonists, we describe here the medicinal chemistry approach leading to the development of CB(2) receptor inverse agonists with a 4-oxo-1,4-dihydropyridine scaffold. The compounds reported here show high affinity and potency at the CB(2) receptor while showing only modest affinity for the centrally expressed CB(1) cannabinoid receptor. Further, we found that the functionality of this series is controlled by its C-6 substituent because agonists bear a methyl or a tert-butyl group and inverse agonists, a phenyl or 4-chlorophenyl group, respectively. Finally, in silico studies suggest that the C-6 substituent could modulate the conformation of W6.48 known to be critical in GPCR activation.