Assessing the Potential Synergistic/Antagonistic Effects of Citrinin and Cannabidiol on SH-SY5Y, HepG2, HEK293 Cell Lines, and Human Lymphocytes

Abstract

The increasing use of Cannabis sativa products for medicinal, dietary, and recreational purposes has raised concerns about mycotoxin contamination in cannabis and hemp. Mycotoxins persist in these products' post-processing, posing health risks via multiple exposure routes. This study investigated cytotoxic and genotoxic interactions between cannabidiol (CBD) and the mycotoxin citrinin (CIT) using human cell models: SH-SY5Y, HepG2, HEK293, and peripheral blood lymphocytes. IC₅₀ values and membrane disruption were initially assessed, followed by an evaluation of genotoxicity in lymphocytes using the Comet Assay and Cytokinesis Blocked Micronucleus Cytome Assay. Obtained findings demonstrate that cell-type sensitivity varied across treatments, with combined CBD and CIT exposure exhibiting distinct interactions. Lactate dehydrogenase (LDH) release remained minimal, suggesting cytotoxicity did not stem from membrane disruption but likely involved intracellular pathways. In lymphocytes, CBD alone produced negligible cyto/genotoxic effects and weak antiproliferative responses, whereas CIT displayed clear toxic impacts. DNA damage indicates that CIT may induce genome instability through indirect mechanisms rather than direct DNA interaction, with evidence of potential aneuploidic effects from the CBMN Cyt Assay. Combined exposure led to a reduction in CIT-induced DNA and cytogenetic damage, suggesting CIT's potential interference with the beneficial properties of CBD. These results provide a foundation for further toxicological assessments and highlight the necessity of standardized mycotoxin monitoring in cannabis-derived products.

Keywords: Cannabis sativa; cell viability; contaminants; cytotoxicity; genotoxicity; hemp; moulds; mycotoxins.

Figure 1

Dose-dependent cytotoxicity and IC₅₀ values of cannabidiol (CBD) and citrinin (CIT) on SH-SY5Y, HepG2 and HEK293 cells after 24 h exposure. Experimental data are presented as a mean (±SE) of at least three experiments.

Figure 2

Viability of SH-SY5Y, HepG2 and HEK293 cells after 24 h exposure to cannabidiol (CBD), citrinin (CIT) and their combinations at lowest-observed-adverse-effect level (LOAEL) concentrations (1 and 30 μM for SH-SY5Y and HepG2 and 2 and 0.05 μM for HEK293, respectively). Experimental data are presented as a mean (±SE) of at least two experiments. The results are expressed as percentages of corresponding control, untreated cells, and given as means ± SE. & p < 0.05; * p < 0.0001 vs. untreated control.

Figure 3

Levels of lactate dehydrogenase (LDH) release after 24 h exposure of SH-SY5Y, HepG2 and HEK293 cells after 24 h exposure to cannabidiol (CBD), citrinin (CIT) and their combinations at LOAEL concentrations (1 and 30 μM for SH-SY5Y and HepG2, and (2 and 0.05 μM for HEK293, respectively). Triton (0.08%) was used as the positive control. Experimental data are presented as a mean of percentage of LDH release (±SE) of at least two experiments. * p < 0.0001 vs. untreated control.

Figure 4

DNA damage in lymphocytes estimated by the alkaline comet assay. Lymphocyte cultures were treated for 24 h with citrinin (CIT) at 30 µM (7.50 µg/mL), cannabidiol (CBD) at 1 µM (3.15 µg/mL) and their combination (CIT + CBD, at the same concentrations). Negative controls (NCs) were non-treated lymphocytes. Solvent controls (SCs) were lymphocytes treated with ethanol (final concentration in the culture corresponded to 0.03%). Positive controls were lymphocytes treated for 24 h with bleomycin at 1.25 µg/mL. Six hundred independent comet measurements were carried out per experimental point. Results are expressed as mean/median, interquartile range, and range of measured values. Inter-group comparisons were performed using ANOVA with Tukey’s HSD post hoc test. Differences significant at p < 0.05 are marked with: *—vs. all other experimental groups; a—vs. negative control; b—vs. solvent control; c—vs. CBD; d—vs. CIT + CBD.

Figure 5

Typical lymphocyte nuclei observed under an epifluorescence microscope on microgels prepared for the alkaline comet assay. Non-damaged DNA in the negative control (a), and in the solvent control (b). Citrinin-treated lymphocyte (CIT) with damaged DNA (c). Low DNA damage in a cannabidiol-treated lymphocyte (CBD) (d), and in lymphocytes treated with combination of CIT and CBD (e). Highly fragmented DNA in a lymphocyte treated with bleomycin, positive control (f). Stained with ethidium bromide. Photomicrographs were taken under magnification ×200 using a black and white camera coupled with a computer-based image analysis system (Comet Assay IV, Instem-Perceptive Instruments Ltd., Suffolk, UK).

Figure 6

Photomicrographs of typical features observed on microscope slides prepared using Cytokinesis-Block Micronucleus (CBMN) Cytome Assay on human peripheral blood lymphocytes. Arrow indicates binucleated lymphocytes with (a) nuclear bud (NB) in the citrinin-treated sample (CIT); (b) micronucleus (MN) in the negative control sample; (c) two MNi in the citrinin + cannabidiol-treated sample (CIT + CBD); (d) nucleoplasmic bridge (NPB) in CIT-treated sample. Morphological features of dead cells in the positive control sample: (e) apoptotic cell with nuclear fragmentation; (f) a shift from apoptosis to necrosis; (g) necrotic cell. Typical features of cells scored to determine Cytokinesis-Block Proliferation Index: (h) mononucleated cell, M1 in negative control sample; (i) cell with two nuclei, M2 in negative control sample; (j) cell with three nuclei, M3 negative control sample; (k) cell with four nuclei, M4 in CIT-treated sample. Stained with Giemsa. Photographed at magnification ×1000 with Axiocam 208 color camera on Axiolab 5 microscope (Carl Zeiss Microscopy GmbH, Jena, Germany).

Figure 7

Results of analysis of lymphocyte proliferation in cell cultures treated in vitro for 24 h with citrinin (CIT) at 7.50 µg/mL (30 µM), cannabidiol (CBD) at 3.15 µg/mL (1 µM) and their combination (CIT + CBD, at the same concentrations). Negative controls were non-treated lymphocytes. Solvent controls were lymphocytes treated with ethanol (final concentration in the culture corresponded to 0.03%). Positive controls were lymphocytes treated for 24 h with bleomycin at 1.25 µg/mL.