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More Research Needed to Establish Potential Toxicity of Key Cannabinoids

by Jack Coles
Published: Nov 07, 2018   
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Although cannabis is sometimes purported to be less carcinogenic than tobacco, evidence has emerged that the former has its own share of harmful constituents. Researchers from the University of Campania, Italy, and the Medical University of Vienna, Austria, recently found that two active ingredients in cannabis extract – cannabidiol (CBD) and cannabidivarin (CBDV) – had potentially carcinogenic effects.

Cannabidiol and cannabidivarin are two aromatic compounds that differ only by an ethyl group on the terminal carbon chain. Although structurally similar to tetrahydrocannabinol (THC), these compounds exhibit no psychoactive properties. Instead, CBD is used as a prescription medicine in various countries to manage neurological disorders, and CBDV is being developed as an anticonvulsant in adults. This new evidence may be cause for concern.

CBD and CBDV are naturally-occurring compounds and as such there are fewer safety testing requirements that need to be completed prior to sale. They occur naturally in small concentrations in unrefined hemp and cannabis oil, and in larger amounts in Cannabis sativa and C. indica leaf matter.

Some research has been carried out on CBD’s toxicity, but carcinogenicity and genotoxicity are noticeably lacking. Only a handful of experiments carried out on rodents in the 1980s are available in the literature. These studies suggested that CBD has some genotoxic properties (i.e. able to cause damage to chromosome structure), and there are no studies of this kind at all on CBDV.

Assessing the toxicity of CBD and CBDV

The micronucleus test is a measure of genotoxicity used extensively in toxicology. It works by exposing cells to a test chemical and counting the percentage of them that have formed a micronucleus – a structure that arises when chromosomes are damaged and improperly separated during mitosis. The higher the percentage, the more carcinogenic the chemical. Cells can also develop nucleoplasmic bridges and/or nuclear buds, which are also indicators of DNA damage, and these were also measured in the research.

These experiments were carried out using a hepatic cell line (Hep-G2) exposed to CBD or CBDV dissolved in methanol, or to methanol alone (vehicle control), for 3 hours. The highest concentration used was 2mM, which approaches the upper limits of CBD levels in human plasma after smoking cannabis. It should be noted that methanol by itself has no known carcinogenic properties.

The percentage of cells with micronuclei increased in line with escalating CBD and CBDV concentrations. At 2mM over half of the cells had developed micronuclei; similar levels to that caused by the positive control, the genotoxic compound (and chemotherapy agent) cyclophosphamide. Additionally, around 45% of cells developed nuclear buds and 15% developed nucleoplasmic bridges, although there was no indication how many cells had two or more of these features. The proportion of necrotic and apoptotic cells also increased with CBD and CBDV exposure. The negative and vehicle controls both showed values of approximately 5% in each category, and the differences between these and the test groups were statistically significant at concentrations over 0.66 mM.

Another experiment used was the comet assay, also known as single cell gel electrophoresis. This uses an electrical charge to draw DNA out of a well and through some agarose. Undamaged DNA doesn’t move, but fragmented pieces will travel through and create a “comet” or “tail”. The damage is quantified using the percentage of DNA that is in the tail versus the total DNA. These tests were carried out on both the liver-derived Hep-G2 cells and epithelial TR146 cells. 

Like the micronucleus tests, the comet assay showed that increasing concentrations of CBD and CBDV caused an increase in DNA damage. Hep-G2 cells exposed to CBD for 3 hours had more DNA damage than with CBDV, but at 24 hours CBD damage had decreased and CBDV damage had increased. TR146 cells also had increased DNA tails after 3 hours, and adding liver homogenate to these almost doubled the percentages; this indicates that the metabolites of CBD and CBDV could be more genotoxic than the original compounds.

Finally, a modified version of the comet assay was carried out where the cells were treated with CBD or CBDV for three hours, and then had the nuclei exposed to formamidopyrimidine DNA glycosylase (FPG). This enzyme destroys bases that have been oxidized in some way, causing the DNA to break. This allows the measurement of damaged bases, which again increased when the concentration of CBD and CBDV was increased.

The research suggests that both compounds may cause direct damage to genetic material and might be dangerous to human health. It should be noted, however, that the experiments used cell lines in media and as such might not be representative of physiological conditions. Additionally, both cell lines are derived from tumors, which can have different metabolic processes when compared to normal, healthy human cells.

The potential toxicological mechanisms of CBD and CBDV

The authors indicate that their research suggests CBD and CBDV cause damage to DNA by oxidizing it, which is unusual given its supposed anti-oxidant properties. Additionally, the experiments with liver homogenate show that the metabolites could be more dangerous than the original compounds, which indicates that any medical use of CBD and CBDV could benefit from modifying their chemical structure to prevent their metabolism into these toxins.

CBD and CBDV are not the only compounds in cannabis that have been shown to be potentially carcinogenic. Cannabis smoke, like tobacco smoke, contains dangerous polycyclic aromatic hydrocarbons that can also damage cellular DNA and lead to mouth and lung cancer. Additionally, THC has been shown to have immunosuppressive effects, which could expose users to additional risk of cancer as the body’s anti-tumorigenic responses could be dampened.

The research implies that oral or topical ingestion of CBD and CBDV-containing oils (as opposed to smoking cannabis) could certainly be safer due to the reduced number of combustion-derived carcinogens. That said, the oral and dermal bioavailability of CBD is reduced when compared to that of pulmonary bioavailability. People taking CBD on prescription are advised to talk to their doctor if they have any concerns.

Overall, as the researchers themselves said, there needs to be far more research carried out on these compounds before their toxicity can be fully elucidated. Previous research on CBD showed that it is well tolerated by human physiology despite the potential genotoxic effects.


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