Cannabinol in the spotlight: Toxicometabolomic study and behavioral analysis of zebrafish embryos exposed to the unknown cannabinoid.

Cannabinol (CBN) is a degradation product of the cannabis metabolite Δ9-tetrahydrocannabinol. The CBN concentration in cannabis leaves ranges between 0.1 and 1.6% (w/w of dry weight); it increases as the plant ages and its formation is affected by the storage conditions. As CBN has not been extensively studied so far, the need to examine its impact in vivo is imperative due to the increasing use of cannabis globally. In the study herein, the CBN toxicity, effects on heart physiology, morphological malformations, behavioral changes and alterations in metabolic pathways of zebrafish larvae upon CBN exposure to sublethal concentrations were examined. The LD50 value was estimated at 1.12 mg/l. At the same time, malformations in zebrafish larvae increased significantly in a dose-dependent manner and exposure to CBN concentrations greater than 0.75 mg/l provoked abnormalities like pericardial edema, yolk sac anomalies and tail bending. Concentrations above this threshold resulted in elongated and shorter in width hearts and in separation of ventricle from atrium. The total movement distance and velocity were increased in dark and decreased in light conditions, in a concentration-dependent manner. Our results showed that CBN acts both as a stimulant and a sedative, with larvae to exhibit altered velocity and bradycardia, respectively. The metabolomic analysis revealed alterations mainly to amino acids, which are related to acute toxicity and hint towards systemic metabolic and neuropathophysiological changes. Taken together, our data indicate increased toxic effects as CBN exposure concentration increases, which should be taken into consideration when studying the impact of cannabis on organisms.

Marijuana extracts possess the effects like the endocrine disrupting chemicals.

The progesterone 17alpha-hydroxylase activity, which is one of the steroidogenic enzymes in rat testis microsomes, was significantly inhibited by crude marijuana extracts from Delta(9)-tetrahydrocannabinolic acid (THCA)- and cannabidiolic acid (CBDA)-strains. Delta(9)-Tetrahydrocannabinol, cannabidiol and cannabinol also inhibited the enzymatic activity with relatively higher concentration (100-1000 microM). Testosterone 6beta- and 16alpha-hydroxylase activities together with androstenedione formation from testosterone in rat liver microsomes were also significantly inhibited by the crude marijuana extracts and the cannabinoids. Crude marijuana extracts (1 and 10 microg/ml) of THCA strain stimulated the proliferation of MCF-7 cells, although the purified cannabinoids (THC, CBD and CBN) did not show significant effects, such as the extract at the concentration of 0.01-1000 nM. These results indicate that there are some metabolic interactions between cannabinoid and steroid metabolism and that the constituents showing estrogen-like activity exist in marijuana.

Psychoactive cannabinoids increase mortality and alter acute phase cytokine responses in mice sublethally infected with Legionella pneumophila.

Marijuana contains both psychoactive and nonpsychoactive cannabinoids which have varying effects on the immune response system. Previous studies with delta-9-tetrahydrocannabinol (THC), the major psychoactive component of marijuana, showed that this substance augmented the susceptibility of mice to infection with the opportunistic pathogen Legionella pneumophila. The present study compared the enhancement of Legionella-induced mortality in mice due to two other major of marijuana components, cannabinol and cannabidiol, as well as the synthetic psychoactive cannabinoid CP 55,940. Inbred BALB/c mice, relatively resistant to infection with Legionella, were given the marijuana component 1 day before and 1 day after a sublethal intravenous infection with Legionella. Unlike the effect of THC, an 8 mg/kg dose of either cannabinol or cannabidiol did not affect mortality of the mice sublethally infected with Legionella. Mice given a 16 mg/kg dose of these components of marijuana, however, showed a slight to moderately increased mortality following the sublethal infection with Legionella. In contrast, a dose of 6 mg/kg of the synthetic psychoactive cannabinoid CP 55,940 given 1 day before and 1 day after infection with Legionella resulted in about 50% of the animals dying, the same level of augmentation of lethality induced by THC. Liver, spleen, and lung tissues were removed from the surviving mice 24 hr after the second THC injection and tested for the presence of viable Legionella using a standard CFU assay. The mice injected with THC before and after infection showed significantly higher levels of bacteria in their lung than mice that were not given any cannabinoid but were infected sublethally with the Legionella. Mice injected with the other cannabinoids, including the synthetic cannabinoid, showed a much smaller increase in the number of Legionella in their lung when infected with Legionella and treated with the drug. The number of bacteria recovered from the kidney and liver of the mice regardless of treatment with a cannabinoid, including with THC, did not show significant changes. RNA isolated from the spleen of the THC- and Legionella-treated animals was examined to determine at the molecular level whether acute phase pro-inflammatory cytokines (i.e., IL-1, IL-6 and TNF-alpha) were altered following drug treatment and infection, since previous studies had shown there were increased serum levels of these cytokines in the mice. It was found that the mRNA levels for IL-1 remained generally constant regardless of whether the infected animals were treated with a cannabinoid. However, the mRNA level for IL-6 was markedly increased following treatment of the infected animals with THC, suggesting the possible involvement of this pro-inflammatory cytokine in increased mortality. The mRNA level for TNF-alpha was generally low and not significantly altered in the drug treated animals. Mice given other cannabinoids, including cannabinol and cannabidiol, as well as the synthetic CP 55,940, showed no significant change in the level of mRNA for any of the cytokines tested.

Chronic marijuana smoke exposure in the rhesus monkey. IV: Neurochemical effects and comparison to acute and chronic exposure to delta-9-tetrahydrocannabinol (THC) in rats.

THC is the major psychoactive constituent of marijuana and is known to produce psychopharmacological effects in humans. These studies were designed to determine whether acute or chronic exposure to marijuana smoke or THC produces in vitro or in vivo neurochemical alterations in rat or monkey brain. For the in vitro study, THC was added (1-100 nM) to membranes prepared from different regions of the rat brain and muscarinic cholinergic (MCh) receptor binding was measured. For the acute in vivo study, rats were injected IP with vehicle, 1, 3, 10, or 30 mg THC/kg and sacrificed 2 h later. For the chronic study, rats were gavaged with vehicle or 10 or 20 mg THC/kg daily, 5 days/week for 90 days and sacrificed either 24 h or 2 months later. Rhesus monkeys were exposed to the smoke of a single 2.6% THC cigarette once a day, 2 or 7 days a week for 1 year. Approximately 7 months after the last exposure, animals were sacrificed by overdose with pentobarbital for neurochemical analyses. In vitro exposure to THC produced a dose-dependent inhibition of MCh receptor binding in several brain areas. This inhibition of MCh receptor binding, however, was also observed with two other nonpsychoactive derivatives of marijuana, cannabidiol and cannabinol. In the rat in vivo study, we found no significant changes in MCh or other neurotransmitter receptor binding in hippocampus, frontal cortex or caudate nucleus after acute or chronic exposure to THC. In the monkey brain, we found no alterations in the concentration of neurotransmitters in caudate nucleus, frontal cortex, hypothalamus or brain stem.(ABSTRACT TRUNCATED AT 250 WORDS)

Maternal cannabinoid exposure. Effects on spermatogenesis in male offspring.

Maternal exposure to cannabinoids influenced spermatogenesis and fertility in their male offspring examined at 60-80 days of age. Approximately 20% less spermatozoa were found in males whose mothers had received either the non-psychoactive cannabinol (CBN) or cannabidiol (CBD) on day 1 postpartum. Males exposed to the major psychoactive component of marihuana, delta 9-tetrahydrocannabinol (THC) appeared to have spermatozoa in number comparable to controls. This finding may be consistent with the additional observation that CBN or CBD, but not THC, reduced the percentage of successful impregnations by cannabinoid-exposed males. However, males exposed to each of these cannabinoids produced significantly less live offspring compared to controls. Plasma levels of testosterone and luteinizing hormone (LH) were reduced significantly in mice exposed to THC on day 12 of gestation, while testicular weight was reduced in adult mice exposed either on day 12 of gestation to CBD or on day 1 post-partum to THC. These results indicate that perinatal exposure to psychoactive and non-psychoactive components of marihuana can produce long-term disruption of testicular function including the spermatogenic as well as the steroidogenic components.

Perinatal cannabinoid exposure: effects on hepatic cytochrome P-450 and plasma protein levels in male mice.

Maternal exposure to the major psychoactive delta 9-tetrahydrocannabinol (THC), or to the nonpsychoactive cannabinol (CBN) or cannabidiol (CBD) on day 12 of gestation, or on day 1 postpartum, affected the concentrations of hepatic cytochromes P-450 in adult male offspring. Levels of P-450 were significantly increased in adult males prenatally exposed to cannabinoids, but were reduced after postnatal exposure. The response to exogenous testosterone was also differentially affected by perinatal cannabinoid exposure, with reduced plasma androgen in males prenatally exposed to THC, but increased levels of hormone in mice exposed postnatally to THC or CBN. There was a concomitant decrease in plasma albumin and increased gamma-globulin in adult males postnatally exposed to CBN. Beta-globulin levels were also significantly increased in adult males exposed to cannabichromene (CBC) on day 1 postpartum. Cannabinoid exposure during perinatal periods of development exert effects on hepatic function, plasma androgen levels, and on the immune system. These effects may reflect the ability of perinatal cannabinoid exposure to interfere with androgen-mediated processes of differentiation.

Effects and interactions of natural cannabinoids on the isolated heart.

A Langendorff perfused rat heart preparation was designed to process dose-response effects of cardioactive drugs on rate, coronary flow, and supraaortic differential pressure (delta P; an index of cardiac performance). In this preparation, delta 9- -tetrahydrocannabinol (THC) 2 X 10(-6) M to 10(-5) M induces in the isolated perfused rat heart a biphasic increase in rate (maximal at 8 X 10(-6) M). Tachycardia is associated with decreases in (delta P) and no change or decreased coronary flow. Cardiac toxicity is observed with 3 X 10(-5) M. Cannabidiol (CBD) at concentrations of 9 X 10(-6) M to 10(-4) M has limited effect on rate while increasing delta P and coronary flow. Cannabinol (CBN) 8 X 10(-6) M to 3 X 10(-4) M depresses rate and delta P while coronary flow remains constant. Simultaneous equimolar administration of THC with CBD antagonizes or mitigates the cardiac effects of THC on rate, delta P, and coronary flow.

Intraocular pressure, ocular toxicity and neurotoxicity after administration of cannabinol or cannabigerol.

Cannabinol or cannabigerol was administered to cats topically in doses of 250, 500 and 1000 micrograms as a single drop or chronically via osmotic minipumps (20 micrograms hr-1) over a period of 9 days. While cannabinol had a modest effect on intraocular pressure after a single dose, it caused a more significant reduction in ocular tension during chronic administration. Cannabigerol had similar effects, but the magnitude of response to its chronic administration was greater. Cannabinol but not cannabigerol caused conjunctival erythema and hyperemia. After systemic administration of cannabinol (20, 40 or 80 mg kg-1) to rats, 8-13 Hz polyspike discharges appeared in the electrocorticogram during wakefulness and during rapid eye movement sleep episodes. Cannabigerol (10, 30 and 100 mg kg-1) lacked this effect. These results indicate that chronic administration of these cannabinoids lowers ocular tension considerably. Like marihuana and delta-9-tetrahydrocannabinol, cannabinol produced both ocular toxicity and neurotoxicity. As cannabigerol lacked these toxicities, it appears that the ocular hypotensive effect of this cannabinoid is somewhat dissociable from both the adverse central and ocular effects accompanying marihuana intake.

Early cannabinoid exposure influences neuroendocrine and reproductive functions in male mice: I. Prenatal exposure.

Maternal exposure to delta 9-tetrahydrocannabinol (THC), the major psychoactive constituent in marihuana, or to the non-psychoactive cannabinol (CBN) or cannabidiol (CBD) alters endocrine functions and concentrations of brain biogenic amines in their male offspring. Prenatal CBN exposure on day 18 of gestation resulted in decreased plasma FSH levels, testicular testosterone (T) concentrations, and seminal vesicles weights, but increased plasma levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) post-castration in adulthood. Prenatal exposure to THC significantly enhanced the responsiveness of the testes to intratesticular LH injection in vivo and tended to increase human chorionic gonadotropin (hCG)-stimulated T production by decapsulated testes in vitro. In the CBN-exposed mice, hCG-stimulated T production was enhanced, while CBD exposure had no effect. Prenatal THC exposure altered the negative feedback effects of exogenous gonadal steroids in castrated adults, with lower plasma T and FSH levels after 20 micrograms T than in castrated controls. In contrast, CBD-exposed mice had higher levels of LH in plasma post-castration. In CBN-exposed adults, two weeks post-castration the concentration of norepinephrine (NE) and dopamine (DA) in hypothalamus and remaining brain were reduced, while levels of serotonin (5-HT) and its metabolite, 5-HIAA, were elevated compared to that in castrated OIL-controls. Prenatal CBD-exposure also reduced NE and elevated 5-HT and 5-HIAA, but did not affect DA levels post-castration. Concentrations of brain biogenic amines were not influenced by prenatal THC exposure in the present study. A single prenatal exposure to psychoactive or non-psychoactive components of marihuana results in long term alterations in the function of the hypothalamo-pituitary-gonadal axis. Changes in the concentrations of brain biogenic amines may be related to these effects of prenatal cannabinoids on endocrine function in adult male mice.

Early cannabinoid exposure influences neuroendocrine and reproductive functions in mice: II. Postnatal effects.

Maternal exposure to delta 9-tetrahydrocannabinol (THC), the major psychoactive component in marihuana, or to the non-psychoactive cannabinol (CBN) or cannabidiol (CBD) alters male reproductive functions and brain biogenic amines in male and female offspring. Postnatal exposure to THC or CBN reduced body weights, while testicular weights were lower in CBD-exposed mice. Testicular testosterone (T) levels were also lower in CBN- and CBD-exposed animals. Postnatal cannabinoid exposure reduced plasma luteinizing hormone (LH) levels in intact and castrated adults. Although basal T production in vitro was not affected by postnatal cannabinoid exposure, testes from CBD-exposed males were more responsive to gonadotropin stimulation. In contrast, in vivo responsiveness to intratesticular human chorionic gonadotropin (hCG) administration was significantly reduced in THC- and CBD-exposed males. Pituitary weights and their basal LH production in vitro was higher in THC- or CBN-exposed mice. Pituitaries from cannabinoid-exposed males were less responsive to LH releasing hormone (RH) stimulation, however, hypothalamic LHRH content was significantly higher in the THC-exposed males. Hypothalamic dopamine (DA) levels were significantly lower in CBN-exposed castrated mice, compared to castrated controls. The reduction in hypothalmic norepinephrine (NE) in THC- and CBN-exposed castrates after alpha-methylparatyrosine (alpha-MPT) was significantly less than that observed in control castrates. Hypothalamic DA levels were depleted to a greater extent in CBD-exposed males. Brain levels of serotonin (5-HT) and 5-HIAA were significantly higher in castrated, than in intact THC-exposed males. In ovariectomized CBN-exposed females, hypothalamic NE levels were lower, while the alpha-MPT-induced depletion of NE was less in CBD-exposed, compared to control females. Levels of 5-HT were lower only in THC-exposed females. Plasma levels of LH were significantly higher in CBN-exposed, while plasma levels of FSH were reduced in THC- and CBD females. Maternal exposure to psychoactive or non-psychoactive cannabinoids on the day of parturition results in long term alterations in neuroendocrine function in male and female offspring. It is possible that the observed alterations in biogenic amines may mediate the effects of cannabinoids on pituitary and gonadal function.

Fetal testosterone in mice: effect of gestational age and cannabinoid exposure.

The effect of increasing gestational age and maternal exposure to cannabinoids on body weight, ano-genital distance and androgen concentration in fetal mice was examined. Body weight increased in both male and female fetuses from days 16 to 18 (the presence of a vaginal plug was considered to indicate day 1 of pregnancy), while ano-genital distances tended to increase faster in male than in female fetuses. The concentration of testosterone increased with age in fetuses of either sex. However, at day 16, there was a significant influence of fetal sex on testosterone concentration with two non-overlapping distribution, one above and one below 300 pg/g fetal tissue, correlating with male and female gender respectively. After day 16, male fetuses tended to have higher testosterone concentrations, but some values obtained in females did overlap. Treatment of female mice with delta 9-tetrahydrocannabinol, the main psychoactive ingredient of marihuana, from days 12 to 16 pregnancy caused a significant (P less than 0.01) increase in fetal deaths in utero. Cannabinol treatment had no effect on this parameter, but reduced body weight (P less than 0.02) in female fetuses, and increased ano-genital distance (P less than 0.05) in male fetal mice. The concentrations of testosterone and dihydrotestosterone were reduced in male but not in female fetuses. The results indicate that exposure to psychoactive or non-psychoactive constituents of marihuana suppresses testosterone levels in fetal as well as in immature and adult mice, as we have previously reported. Thus, maternal exposure to cannabinoids may interfere with the process of sexual differentiation in their male offspring as a result of decreased fetal androgen production.

In vivo cytogenetic effects of cannabinoids.

The cytogenetic effects of subacute oral administration of delta-9-tetrahydrocannabinol, cannabidiol, cannabinol, and placebo were assessed in 27 subjects in a prospective double-blind study. Both conventional chromosome aberration analysis and sister chromatid exchange were considered. A comparison group of 5 nondrug users was also investigated. The aneuploidy frequency decreased significantly following delta-9-tetrahydrocannabinol administration. By contrast, the frequencies of chromosome aberrations increased slightly for all three drug groups and decreased in the placebo and comparison groups although none of the predrug versus postdrug comparisons were statistically significant. No increase in the sister chromatid exchange frequency was found in any of the groups when predrug versus postdrug comparisons were made. The present study is the fourth short term prospective investigation failing to demonstrate a statistically significant increase in chromosome damage due to cannabinoid usage. However, it must be kept in mind that to date only gross chromosomal aberrations have been identified so that the more subtle chromosomal as well as molecular genetic damage has not been ruled out.

Effects of cannabinoids on sperm morphology.

Sperm morphology was studied in hybrid mice of genotype (C57BL X C3H)F1 following treatment with specific cannabinoids. Mice were treated for 5 consecutive days with the specific cannabinoid; 35 days after the last treatment, epididymal sperm were scored in the light microscope and assessed in the scanning electron microscope. The animals treated with delta9-tetrahydrocannabinol (delta9-THC) and cannabinol (CBN) had a statistically higher incidence of abnormal sperm than the controls. The incidence of abnormal sperm in the animals treated with cannabidiol (CBD) was not statistically different from the control value. The relative toxicity of the cannabinoids in these studies was delta9-THC greater than CBN greater than CBD. Normal sperm have a smooth kidney-shaped head with a prominent hook; abnormal sperm have shapes which include heads without hooks, banana-shaped heads, amorphous heads and folded heads.

[Organic lesions after application of hashish in rats and rabbits (author's transl)]. / Organveränderungen bei Ratten und Kaninchen nach Haschischapplikation.

Toxic lesions were found in liver, kidney and brain of rats and rabbits after oral application of hashish for 7 or 30 days. The brain tissue showed hyperemia and edema. The cerebral cortex, brain stem and cerebellum contained ganglion cells with eccentric nuclei and an icreased number of nucleoli. Different stages of cell lesions could be distinguished by nuclear vacuoles, pyknosis and karyolysis. The cytoplasma was loosened and showed numerous vacuoles. In the kidney we found, besides interstitial hemorrhage, cloudy swelling of the proximal tubules with vacuolous degeneration and nuclear pyknosis and karyolysis in all stages. The glomeruli were often enlarged with thickening of Bowman's capsule and surrounding cellular infiltration with cell fragments. The liver cells showed irregularities of nuclei and vacuolated nuclei, plasma vacuoles and cell necrosis. Infiltrations of round cells at the portal-biliary areas were usually combined with an activation of the Kupffer cells and intracellular bile stasis.