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. Author manuscript; available in PMC: 2023 Apr 24.
Published in final edited form as: Toxicol Sci. 2023 Feb 17;191(2):227–238. doi: 10.1093/toxsci/kfac131

Cannabidiol-induced transcriptomic changes and cellular senescence in human Sertoli cells

Yuxi Li 1, Xilin Li 2, Patrick Cournoyer 3, Supratim Choudhuri 4, Xiaozhong Yu 5, Lei Guo 1, Si Chen 1,*
PMCID: PMC10123764  NIHMSID: NIHMS1887839  PMID: 36519830

Abstract

Cannabidiol (CBD), one of the major cannabinoids in the plant Cannabis sativa L., is the active ingredient in a drug approved for the treatment of seizures associated with certain childhood-onset epileptic disorders. CBD has been shown to induce male reproductive toxicity in multiple animal models. We previously reported that CBD inhibits cellular proliferation in the mouse Sertoli cell line TM4 and in primary human Sertoli cells. In this study, using a transcriptomic approach with mRNA sequencing analysis, we identified molecular mechanisms underlying CBD-induced cytotoxicity in primary human Sertoli cells. Analysis of differentially expressed genes (DEGs) demonstrated that DNA replication, cell cycle, and DNA repair were the most significantly affected pathways. We confirmed the concentration-dependent changes in the expression of key genes in these pathways using real-time PCR. mRNA-sequencing showed upregulation of a group of genes tightly associated with senescence associated secretory phenotype (SASP) and with the activation of the p53 signaling pathway, a key upstream event in cellular senescence. Prolonged treatment of 10 μM CBD induced cellular senescence, as evidenced by the stable cessation of proliferation and the activation of senescence associated β-galactosidase (SA-β-gal), two hallmarks of senescence. Additionally, using real-time PCR and Western blotting assays, we observed that CBD treatment increased the expression of p16, an important marker of cellular senescence. Taken together, our results show that CBD exposure disturbs various interrelated signaling pathways and induces cellular senescence in primary human Sertoli cells.

Keywords: cannabidiol, primary human Sertoli cells, mRNA sequencing, DNA replication, DNA repair, cell cycle, cellular senescence, male reproductive toxicity

Introduction

The use of cannabis-containing drugs and consumer products has increased dramatically in recent years, driven by their perceived health benefits and increased availability. The cannabis plant, Cannabis sativa L., contains more than 100 cannabinoids (Aizpurua-Olaizola et al., 2016; Rock and Parker, 2021). One of the major cannabinoids, cannabidiol (CBD), is a non-intoxicating compound that has been investigated for a wide range of pharmacological effects (Huestis, 2007). The U.S. Food and Drug Administration (FDA) approved Epidiolex®, a CBD-based oral solution, to treat seizures associated with two rare and severe forms of childhood-onset epileptic disorders (Lennox-Gastaut syndrome and Dravet syndrome) and tuberous sclerosis complex.

The oral administration of CBD in monkeys, rats, and mice has been linked to male reproductive toxicity, such as smaller testicles, fewer germ and Sertoli cells, lower fertilization rates, and decreased levels of sex hormones (Carvalho et al., 2020; Carvalho et al., 2018; Dalterio and deRooij, 1986; Rosenkrantz and Esber, 1980; Rosenkrantz et al., 1981). CBD treatment in rodents has also been associated with impaired sexual behavior and abnormal sperm morphology (Carvalho et al., 2020; Patra and Wadsworth, 1991). A recent study reported that long-term (2–4 months) exposure to cannabis vapor (6.7% Δ9-tetrahydrocannabinol and 0.02% CBD) in mice caused a reduction of sperm count and motility and disrupted spermatogenetic progression (Shi et al., 2022). In light of these observations, it is important to evaluate male reproductive toxicity of CBD in human-relevant models and to study its underlying mechanisms.

Sertoli cells are essential for the development of testis during embryonic stages and spermatogenesis in adulthood (Petersen and Soder, 2006). Sertoli cells enclose germ cells and provide physical and nutritional support to their growth and maturation; therefore, the number of Sertoli cells influences testis size and sperm output (Orth et al., 1988; Sharpe et al., 2003). Another major function of Sertoli cells is to form a blood-testis barrier that governs the environment for the development of haploid germ cells and prevents toxins from entering the seminiferous tubules (Petersen and Soder, 2006). The proliferation of Sertoli cells in humans occurs during fetal and neonatal phases and during the peripubertal period (Sharpe et al., 2003). Due to its importance in male reproductive physiology, a decrease in Sertoli cell number results in a reduction in the quality and quantity of sperm, which is linked to the malfunctioning of the testis (Sharpe et al., 2003). Sertoli cells are reported to be one of the targets in male reproductive system for toxins (Ni et al., 2019; Petersen and Soder, 2006). In a previous study, we reported that a 24-h CBD treatment inhibited cellular proliferation and induced G1 cell cycle arrest in both human and mouse Sertoli cells (Li et al., 2022), which correlates with previous in vivo studies where CBD decreased the number of Sertoli cells in mice during adolescence (Carvalho et al., 2018).

Cell cycle arrest often occurs when the surveillance system detects an abnormality in proliferative cells. Growth arrest ensures that the cells have time to repair the cellular damage before progressing. Failure to repair the damage leads to multiple biological processes, including senescence, an anti-proliferative response (Childs et al., 2014). Long-term cell cycle arrest is a typical feature of cellular senescence that was first identified in cultured human fibroblasts characterized by an exhaustion of replicative potential (Hayflick, 1965; Hayflick and Moorhead, 1961). Later, in both in vitro and in vivo studies, cellular senescence was reported in different cell types, triggered by developmental signals or intrinsic and extrinsic stimuli (Kumari and Jat, 2021; Petrova et al., 2016). Induction of p53, p21, or p16 is recognized as an upstream event of cellular senescence (Kumari and Jat, 2021; McConnell et al., 1998). In addition, a major feature of senescent cells is an increased activity of senescence-associated β-galactosidase (SA-β-gal), which is also the most widely used marker of cellular senescence in mammalian cells (Dimri et al., 1995). Moreover, distinct intracellular alternations occur in senescent cells, such as a complex pro-inflammatory secretory phenotype called the Senescence-Associated Secretory Phenotype (SASP) (Ou et al., 2021). Specifically, a series of secretory proteins, including interleukins (IL), chemokines, growth factors, and proteases constitute the SASP secretome. SASP is the key regulator in cellular senescence that occurs in both normal and disease states (Coppe et al., 2010). A recent in vitro study using testicular peritubular cells from non-human primates reported that cellular senescence is associated with impaired protein secretion and reduced contractility in peritubular cells (Stöckl et al., 2020).

We previously reported that CBD inhibited cellular proliferation in primary human Sertoli cells. In this study, to uncover the molecular mechanisms underlying CBD caused by the inhibition of cell proliferation, we determined the transcriptomic profiles altered by CBD in primary human Sertoli cells using mRNA-sequencing (mRNA-seq) technique. Subsequently, we selected and validated the genes affected by CBD in a concentration-dependent manner and analyzed the related biological pathways. Finally, we investigated if treating human Sertoli cells with CBD led to cellular senescence.

Materials and Methods

Test chemicals

CBD (Batch # NQSS1951, stated purity 100%) was purchased from Purisys (Athens, GA). The identity and purity were confirmed by in-house 1H-nuclear magnetic resonance and mass spectral analyses. Dimethyl sulfoxide (DMSO) was purchased from MilliporeSigma (St. Louis, MO) and used as the vehicle. CBD was dissolved in DMSO and stored as 1,000 × stock solution at −20 °C.

Cell cultures

Primary human Sertoli cells were isolated from an 18-week male donor and purchased from ScienCell Research Laboratories (Carlsbad, CA). Cells were cultured in Sertoli cell medium supplemented with 5% fetal bovine serum, 1% Sertoli cell growth supplement, and 1% antibiotic solution (ScienCell Research Laboratories) at 37 °C in a humidified atmosphere with 5% CO2. All culturing vessels were pre-coated with poly-L-lysine (2 μg/cm2, MilliporeSigma) overnight at room temperature and rinsed twice with sterile water. A total of 3 × 105 of human Sertoli cells was seeded in 100 mm tissue culture dishes and subcultured by trypsinization (0.05% trypsin-EDTA solution; ScienCell Research Laboratories) every three days for up to 10 passages.

RNA isolation

To isolate total RNA, human Sertoli cells were seeded in pre-coated 6-well plates at a density of 1.7 × 105 cells/well and cultured approximately for 24 h to allow cells to attach to the surface of vessels. The cells were treated with 7–10 μM CBD or the vehicle (0.1% DMSO) for 24 h. The concentration selection and coverage were based on the results of our previous study, which showed that 7 μM CBD did not have an inhibitory effect on proliferation of human Sertoli cells, whereas 10 μM CBD reduced the proliferation rate of human Sertoli cells to 65.2% of the control after a 24-h treatment (Li et al., 2022). Total RNA was isolated using a RNeasy Mini kit (Qiagen, Valencia, CA). An RNA 6000 Nano kit (Agilent Technologies, Santa Clara, CA) and an Agilent Technologies 2100 Bioanalyzer (Agilent Technologies) were used to measure the purity and quality of RNA. RNA samples with the RNA integrity number (RIN) > 9 were used for subsequent mRNA-seq (Poly-A) and real-time PCR analyses.

Library construction, sequencing, and bioinformatics process

mRNA-Seq library preparation and sequencing were performed at Admera Health, LLC (South Plainfield, NJ). The quality of isolated total RNA samples was assessed using an RNA Tapestation system (Agilent Technologies) and then quantified by Qubit 2.0 RNA HS assay (Thermo Fisher Scientific, Massachusetts). To isolate poly(A)+ transcripts from total RNA samples, paramagnetic beads coupled with oligo d(T)25 were used according to NEBNext® Poly(A) mRNA Magnetic Isolation Module manual (New England BioLabs, Ipswich, MA). Samples were randomly primed (5´ d(N6) 3´ [N=A, C, G, T]) and fragmented based on the manufacturer’s recommendations prior to first strand synthesis. The first strand was synthesized using the Protoscript II Reverse Transcriptase with a longer extension period at 42°C for 30 min. All remaining steps for library construction were followed the manual of NEBNext® Ultra™ II Non-Directional RNA Library Prep Kit for Illumina® (New England BioLabs). The quantity and quality of the final libraries were assessed using Qubit 2.0 (Thermo Fisher Scientific) and TapeStation HSD1000 ScreenTape (Agilent Technologies). The size of the final library was approximately 400 bp with an insert size of about 250 bp. Illumina® 8-nt dual-indices were used and equimolar libraries were pooled based on QC values. Samples were sequenced on an Illumina® NovaSeq S4 (Illumina, San Diego, CA) with a read length configuration of 150 PE for 40 M PE reads per sample (20 M in each direction).

The following bioinformatics pipeline was used for data analysis: FastQC (version v0.11.8) was used to check the quality of reads. Trimmomatic (version v0.38) was applied to remove adaptors and trim low-quality bases (default setting used). STAR Aligner version 2.7.1a was applied to align the reads. Picard tools (version 2.20.4) were used to identify duplicates of mapping. StringTie (version 2.0.4) was applied to assemble the mRNA-Seq alignments into potential transcripts. FeatureCounts (version 1.6.0)/HTSeq was used to count mapped reads for genomic features, such as genes, exons, promoter, gene bodies, genomic bins, and chromosomal locations. DESeq2 (version 1.14.1) was applied to conduct the differential analysis.

Transcriptome concentration-dependency analyses

For the transcriptional concentration-response analyses, BMDExpress software (version 2.30.0507 BETA, 2021) was used after minor modifications to previously described methods (Phillips et al., 2019; Yang et al., 2007). Briefly, after normalization and filtering low expressed genes (mean count =< 1), 21,434 genes were uploaded to the BMDExpress and annotated by Ensembl human genome ID (hg38_ensembl). Williams’ trend tests were used to filter the transcripts that showed a concentration-response pattern (maximum fold change > 2 and adjusted p-value < 0.05). Subsequently, 3,763 transcripts that passed the criteria were further subjected to pathway analysis using DAVID Bioinformatics (https://david.ncifcrf.gov/home.jsp). The DAVID Functional Annotation tool identified 3,128 genes in Homo sapiens; of these, 1,055 genes were mapped onto KEGG pathway database (http://www.kegg.jp/).

Quantitative real-time PCR

Complementary DNA (cDNA) was produced by reverse transcription of 2 μg total RNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA). To measure gene expression levels, real-time PCR assays were performed using FastStart Universal Probe Master (Rox, MilliporeSigma) and the following probes (Thermo Fisher Scientific): E2F1 (Hs00153451_m1), E2F7 (Hs00987777_m1), MCM3 (Hs00172459_m1), PRIM1 (Hs00265388_m1), POLE2 (Hs00160277_m1), POLD1 (Hs01100821_m1), CDC25A (Hs00947994_m1), CDKN1A (Hs00355782_m1), EXO1 (Hs01116190_m1), MRE11 (Hs00967437_m1), NEIL3 (Hs00217387_m1), FANCB (Hs00537483_m1), CDKN2A (Hs00923894_m1), IL-1A (Hs00174092_m1), IL-6 (Hs00174131_m1), and GAPDH (Hs02758991_g1). Assays were run in three technical replicates per experiment under universal cycling conditions (10 min at 95°C; 15 s at 95°C, 1 min 60°C, 40 cycles). The relative expression level of each gene was calculated using the 2−ΔΔCt method and normalized to the expression level of GAPDH.

Western Blot analysis

To extract proteins, human Sertoli cells were plated in pre-coated 100 mm dishes at a density of 1 × 106 cells/dish for 24 h prior to treatment with CBD or the vehicle. After 24 h of CBD treatment, the cells were collected and lysed in RIPA buffer containing Halt™ protease inhibitor cocktail (Thermo Fisher Scientific). The targeted proteins were analyzed by Western blotting as previously described (Li et al., 2021). Primary antibodies targeting phospho-p53 Ser 15 (#9286), p53 (#9282), p21 (#2947), p16 (#92803), and IL-6 (#12153) were obtained from Cell Signaling Technology (Danvers, MA). GAPDH (#5174, Cell Signaling Technology) was used as the internal control. Secondary horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibodies (sc-2357) and anti-mouse IgG (sc-516102) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). AlphaView software (San Jose, CA) was used to quantify the intensity of the protein bands.

Measurement of population doubling

Cell numbers were determined at the designated day for the measurement of population doublings. A total of 3.4 × 104 of human Sertoli cells was seeded into each well of pre-coated 6-well plates in triplicate. Day 0 was defined as when 10 μM CBD or the vehicle (DMSO) was added to medium. DMSO-treated control cells were sub-cultured by trypsinization at 90–95% confluency on Day 3 and then sub-cultured every three days for up to 12 days. Cell numbers were determined at each sub-culture. After counting, the same number (3.4 × 104) of DMSO-treated control cells were re-seeded to new plates with fresh media containing DMSO.

When performing a time course pilot study with CBD-treated cells, we observed that on Days 6–12, the cell number did not exceed the initial seeded number (3.4 × 104) and was not sufficient for re-seeding. As such, cells treated with CBD were kept in their initial plates and CBD-containing media was refreshed every 3 days. On Days 3, 6, 9, and 12, the CBD-treated cells were dissociated by trypsinization and then cell number was determined. The population doubling (PD) between two consecutive time points was calculated based on the formula, PD = log(N2/N1)/log2, where N1 was the cell number at the earlier time point and N2 was the number of cells determined at the latter time point (Kwong et al., 2013; Shay and Wright, 1989). Cumulative population doubling (CPD) at each time point was calculated based on the formula, CPD= PD (A) + PD (B), where PD (A) was the population doubling of the current time point and PD (B) was the population doubling of the earlier time point (Chen et al., 2013).

Measurement of senescence-associated β-galactosidase activity

For the quantitative analysis of SA-β-gal activity, a CellEvent™ senescence green flow cytometry assay kit (Cat. # C10841, Thermo Fisher Scientific) was used. Human Sertoli cells were seeded in pre-coated 6-well plates and treated with CBD or the vehicle for up to 12 days as previously described in the measurement of population doubling section. On Days 3, 6, 9, and 12, cells were collected after trypsinization. Each sample was counted and adjusted to 1 × 105 cells. The cells were washed with 1 × PBS and then resuspended in 100 μL of 4% formaldehyde for fixation. Samples were protected from light and incubated at room temperature for 10 min. Cells were washed with 1% BSA in 1 × PBS and then resuspended in 100 μL of 1:1000 diluted staining solution. Samples were incubated at 37 °C without CO2 for 2 h. After washing and resuspending in 1% BSA in 1 × PBS, the cells were analyzed using a BD FACSCanto II flow cytometer with a 488-nm laser. The stopping gate was set to record 10,000 events.

A SA-β-gal staining assay (#9860, Cell Signaling Technology) was performed to detect the activity of SA-β-gal. After a 12-day treatment with CBD or the vehicle, human Sertoli cells in 6-well plates were washed with 1 × PBS and fixed with 1× Fixative Solution (#9860, Cell Signaling Technology) at room temperature for 10 min. After fixation, cells were washed twice with 1 × PBS and incubated with β-galactosidase staining solution overnight at 37 °C in a dry incubator (no CO2). During the incubation, plates were sealed with Parafilm to prevent evaporation. Images were acquired by Cytation 5 Cell Imaging Reader (BioTek, Winooski, VT) from randomly chosen fields (n>3) and at least 300 cells were examined for each group.

Statistical analyses

Data are presented as the mean ± standard deviation (SD) of three independent experiments. GraphPad Prism 9 (La Jolla, CA) was used for the statistical analyses. One-way analysis of variance (ANOVA) followed by Dunnett’s test was used to determine the difference between the experimental and control groups. Concentration-dependent trends were assessed by linear regression analyses. A p<0.05 was regarded as statistically significant.

Results

CBD treatment repressed the DNA replication pathway in human Sertoli cells

Our previous study demonstrated that inhibition of cellular proliferation and DNA synthesis was the most notable phenotype following CBD treatment in both human and mouse Sertoli cells (Li et al., 2022). In this study, using mRNA-seq analysis, we identified molecular mechanisms underlying CBD-induced cytotoxicity. Total RNA samples were isolated from human Sertoli cells treated with 7–10 μM CBD for 24 h. Based upon William’s trend tests (Phillips et al., 2019), we found that 3,763 out of 21,434 genes demonstrated concentration-dependent changes in expression upon CBD treatment. Subsequently, genes showing concentration-response patterns were mapped onto KEGG database. As shown in Table 1, using an adjusted P-value < 0.1 as the cutoff, 12 KEGG pathways were found to be significantly enriched.

Table 1.

Concentration-dependent differentially transcribed genes enriched pathways significantly altered by CBD treatment in human primary Sertoli cells

No. KEGG pathway ID Adjusted p value Numbers of genes with concentration-dependent changes Percentage of genes in the pathway (%) Fold enrichment
1 hsa03030: DNA replication 7.82E-12 27 0.86 4.89
2 hsa04110: Cell cycle 3.54E-10 52 1.66 2.73
3 hsa03460: Fanconi anemia pathway 7.07E-07 27 0.86 3.32
4 hsa00100: Steroid biosynthesis 4.39E-05 14 0.45 4.56
5 hsa03430: Mismatch repair 4.39E-05 15 0.48 4.25
6 Hsa03440: Homologous recombination 2.45E-04 16 0.51 3.60
7 hsa03410: Base excision repair 0.028 14 0.45 2.77
8 hsa04114: Oocyte meiosis 0.035 31 0.99 1.82
9 hsa04350: TGF-beta signaling pathway 0.043 25 0.80 1.94
10 hsa04115: p53 signaling pathway 0.053 21 0.67 2.04
11 hsa05200: Pathways in cancer 0.061 82 2.62 1.36
12 hsa03420: Nucleotide excision repair 0.079 16 0.51 2.22
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Among these pathways, the DNA replication pathway showed the lowest adjusted P-value, indicating that this pathway exhibited the most significant change in gene expression (Table 1). By analyzing the genes annotated in KEGG pathways and conducting additional literature reviews, we identified 36 genes in the DNA replication pathway that changed after CBD exposure in a concentration-dependent manner (Figure 1A and Supplementary Table S1). Among these genes, CBD treatment downregulated the expression of E2F1 and E2F2 and upregulated the expression of E2F7 (Figure 1A and Supplementary Table S1). The E2F family plays a crucial role in the regulation of cellular proliferation. E2F1 and E2F2 are transcriptional activators that promote the expression of genes involved in S-phase entry, DNA synthesis, and mitosis (Ishida et al., 2001; Ren et al., 2002), whereas E2F7 is a transcriptional repressor that can suppress cellular proliferation (Westendorp et al., 2012). The changes in E2F1 and E2F7 were validated using real-time PCR; the results showed that CBD treatment induced a concentration-dependent decrease in E2F1 and increase of E2F7 transcription (Figure 1B and C).

Figure 1. CBD treatment represses DNA replication pathway in human Sertoli cells.

Figure 1.

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Total cellular RNAs were extracted from human Sertoli cells after a 24 h treatment with DMSO (0 μM) or 7–10 μM CBD. Gene expression profiles (n=4/group) were analyzed by mRNA-seq. (A) A heatmap shows concentration-dependent changes of representative DNA replication-related genes. Relative expression of E2F1 (B), E2F7 (C), MCM3 (D), PRIM1 (E), POLE2 (F), and POLD1 (G) was determined using real-time PCR. The bar graphs represent means ± standard deviation (SD) (n=3). #, significant concentration-related linear trend. *, significantly different from the DMSO control.

As shown in Figure 1A and Supplementary Table S1, the remaining 33 genes that were downregulated by CBD treatment were involved in most steps of DNA replication. For example, minichromosome maintenance (MCM) 2–7 complex unwinds DNA double strands (Bell and Dutta, 2002); MCM8 functions in replication elongation as a DNA helicase (Maiorano et al., 2005); and MCM10, a replication factor, stabilizes and recruits DNA polymerase α to DNA helicase (Perez-Arnaiz et al., 2017; Ricke and Bielinsky, 2004). Primase (PRIM1 and 2) is responsible for synthesizing RNA primers that are required to initiate DNA synthesis (Waga and Stillman, 1998). DNA polymerase α (POLA1–2) and DNA polymerase ε (POLE) synthesize DNA strands immediately after the primase (Waga and Stillman, 1998) and during DNA elongation (Pursell et al., 2007); and DNA polymerase δ (POLD) generates the lagging strand (Nick McElhinny et al., 2008). Using real-time PCR, some of the above noted genes, including MCM3, PRIM1, POLE2, and POLD1, were validated; the results showed concentration-dependent decreases in transcription (Figure 1D–G).

CBD treatment altered the cell cycle pathway in human Sertoli cells

KEGG pathway analysis showed that the cell cycle pathway exhibited the second most significant changes in transcription (Table 1). As shown in Figure 2A and Supplementary Table S2, we observed concentration-dependent changes of some key regulators in the cell cycle pathway, such as cell division cycle (CDC) proteins, serine/threonine kinases, cyclin-dependent kinases (CDKs), checkpoint proteins, stress sensors, CDK inhibitors, and cyclins. The decreases of cyclin D3 (CCND3) and CDK2 genes were consistent with our previous observation that CBD decreased the protein expression of cyclin D3 and CDK2 in both human and mouse Sertoli cells (Li et al., 2022). Using real-time PCR, we confirmed the concentration-dependent changes of two representative genes, cell division cycle 25 A (CDC25A) and cyclin-dependent kinase inhibitor 1A (CDKN1A/p21). When human Sertoli cells were exposed to 10 μM CBD for 24 h, the mRNA level of CDC25A decreased to 9% of the DMSO control (Figure 2B), while the mRNA level of CDKN1A/p21 increased 3.2-fold (Figure 2C). CDC25 phosphatases (CDC25A-C) are the key regulators of the cell cycle, mainly via phosphorylating cyclin E-CDK2 and cyclin A-CDK2 complexes, which promotes the G1 to S transition (Blomberg and Hoffmann, 1999; Boutros et al., 2007). In contrast, CDKN1A/p21 encodes a cyclin-dependent kinase inhibitor that binds and inhibits the activity of cyclins-CDK2 and cyclins-CDK4/6 complexes, which halts the cell cycle progression at G1 phase (Harper et al., 1993; Harper et al., 1995; Kumari and Jat, 2021). The decrease in CDC25A and increase in CDKN1A/p21 transcription are consistent with our previous observation that cells failed to pass G1/S transition point upon CBD treatment (Li et al., 2022).

Figure 2. CBD treatment alters the expression of cell cycle-related genes in human Sertoli cells.

Figure 2.

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(A) A heatmap shows concentration-dependent changes of representative cell cycle-related genes. (B and C) Relative expression of CDC25A (B) and CDKN1A (C) was measured using real-time PCR in CBD-treated human Sertoli cells. The bar graphs show means ± SD (n=3). #, significant concentration-related linear trend. *, significantly different from the DMSO control.

CBD treatment inhibited DNA repair pathways in human Sertoli cells

DNA repair activities are essential to maintain genome integrity. mRNA-seq results and KEGG pathway analysis indicated that CBD treatment inhibited gene expression of central enzymes in various DNA repair pathways, including mismatch repair, homologous recombination-mediated repair, base excision repair, nucleotide excision repair, and Fanconi anemia pathways in human Sertoli cells (Table 1, Figure 3A, and Supplementary Table S3). These pathways play critical roles in repairing mismatches (base substitutions, insertions, and deletions), DNA double-strand breaks, chemically damaged bases, bulky nucleotides, and inter-strand crosslinks (Chatterjee and Walker, 2017). For instance, exonuclease 1 (EXO1) is essential for mismatch repair and DNA double-strand break repair (Li et al., 2019; Qiu et al., 1999). Meiotic recombination 11 (MRE11) is an important nuclease involved in the initiation of double-strand break repair (Stracker and Petrini, 2011). Nei like DNA Glycosylase 3 (NEIL3) is required for the first step of base excision repair (Krokeide et al., 2009). Fanconi anemia complementation group B (FANCB), together with other Fanconi anemia complementation (FANC) family proteins, is assembled into a nucleoprotein complex that is involved in the repair of inter-strand crosslink DNA lesions (Moldovan and D’Andrea, 2009). As shown in Figures 3B–E, concentration-dependent decreases of EXO1, MRE11, NEIL3, and FANCB were observed in both mRNA-seq analysis and real-time PCR validation. After a 24-h treatment, 10 μM CBD reduced the mRNA levels of EXO1, MRE11, NEIL3, and FANCB to 3.9%, 25.2%, 3.1%, and 19.4%, respectively, of the DMSO control (Figures 3B–E). These downregulations suggest that DNA repair activities could be inhibited by CBD in a concentration-dependent manner in human Sertoli cells.

Figure 3. CBD treatment inhibits DNA repair-related genes in human Sertoli cells.

Figure 3.

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(A) A heatmap shows concentration-dependent changes of representative DNA repair-related genes. (B-E) Relative expression of EXO1 (B), MRE11 (C), NEIL3 (D), and FANCB (E) was measured using real-time PCR in CBD-treated human Sertoli cells. The bar graphs show means ± SD (n=3). #, significant concentration-related linear trend. *, significantly different from the DMSO control.

CBD treatment activated p53 signaling pathway in human Sertoli cells

mRNA-seq results and KEGG pathway analysis also revealed that the p53 signaling pathway, a key transduction network that governs multiple cellular processes, was altered in response to CBD treatment (Table 1, Figure 4A, and Supplementary Table S4). As shown in Figure 4A, reported p53 targeted genes (Fischer, 2017), including phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1), sestrin2 (SESN2), stratifin (SFN), G2 and S phase-expressed protein 1 (GTSE1), and ribonuclease P protein component (RPM2), were up- or down-regulated in a concentration-dependent manner by CBD treatment. Furthermore, as shown in Figure 2A, the increases of two well-known p53 targets, CDKN1A/p21 and growth arrest and DNA damage inducible alpha (GADD45A), were observed. Using Western blot analyses, we observed that CBD treatment increased the protein levels of phosphorylated p53 (on serine 15; Ser 15), p53 and p21 (Figures 4B–E), which confirmed further the activation of p53 in CBD-treated human Sertoli cells. Moreover, the increase of E2F7 (Figure 1C) is another indicator of the activation of p53 signaling pathway, because E2F7 has been reported to be a direct target of p53. An increase of E2F7 can further lead to the repression of cell cycle genes (Aksoy et al., 2012; Carvajal et al., 2012); interestingly an increase of E2F7 has been observed during cellular senescence (Aksoy et al., 2012). These findings, including the phosphorylation/activation of p53, upregulation of its target genes of E2F7 (Figures 1A and 1C) and p21, and the cell cycle arrest in G1 phase (Li et al., 2022), suggest that p53-dependent cellular senescence is a possible mechanism underlying CBD’s toxicity. Thus, these findings prompted us to investigate further whether cellular senescence occurred in CBD-treated human Sertoli cells.

Figure 4. CBD treatment activates the p53 signaling pathway in human Sertoli cells.

Figure 4.

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(A) A heatmap shows concentration-dependent changes of representative genes in the p53 signaling pathway. (B-E) Western blot analysis of phospho-p53 (Ser15), p53, and CDKN1A/p21 was conducted in CBD-treated human Sertoli cells. GAPDH was used as an internal control. Representative Western blots are shown in (B). Quantification of relative band intensity is shown in (C-E). The bar graphs represent means ± SD (n=3). The intensity of each protein band was normalized to its individual GAPDH band. *, significantly different from the DMSO control.

CBD induced senescence-associated secretory phenotype

One key characteristic of senescent cells is SASP (Kumari and Jat, 2021). As shown in Figure 5A and Supplementary Table S5, mRNA-seq results indicated that a 24-h CBD treatment increased the expression of multiple SASP-related genes, including interleukins (ILs), chemokines, inflammatory factors, growth factors and regulators, proteases and regulators, soluble ligands, and laminin (Coppe et al., 2010), in a concentration-dependent manner. IL-6 is one of the most prominent cytokines of SASP, and an increase of IL-6 and IL-1A is commonly observed in senescent cells (Coppe et al., 2010). As shown in Figure 5B, the gene expression of IL-1A and IL-6 significantly increased in a concentration-dependent manner. Treatment with 10 μM of CBD for 24 h increased the gene expression of IL-1A and IL-6 41.9- and 36.2-fold, respectively (Figure 5B). Furthermore, an increased protein level of IL-6 was demonstrated using Western blot analysis (Figure 5C). Therefore, the induction of these SASP components indicates the occurrence of cellular senescence upon CBD treatment of human Sertoli cells.

Figure 5. CBD treatment induces SASP-related gene expression.

Figure 5.

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(A) A heatmap shows concentration-dependent changes of representative genes in SASP. (B) Relative gene expression of IL-1A (left panel) and IL-6 (right panel) was measured using real-time PCR in human Sertoli cells after a 24 h CBD treatment. (C) Western blot analysis of IL-6 was conducted in human Sertoli cells after a 24 h CBD treatment. GAPDH was used as an internal control. Representative Western blots are shown in (C, left panel). Quantification of relative band intensity is shown in (C, right panel). The bar graphs represent means ± SD (n=3). #, significant concentration-related linear trend. *, significantly different from the DMSO control.

CBD treatment induced cellular senescence

To examine further that the cells underwent cellular senescence upon CBD exposure, we examined the expression level of a key regulator of senescence, p16, which is crucial to a lasting cell cycle arrest and the maintenance of the senescent state (Kumari and Jat, 2021). Using real-time PCR and Western blotting analyses, we found that a 24-h CBD treatment caused a significant increase of p16 at both gene transcript (Figure 6A) and protein (Figure 6B) levels.

Figure 6. CBD treatment induces cellular senescence phenotypes in human Sertoli cells.

Figure 6.

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(A) Relative gene expression of CDKN2A/p16 was measured using real-time PCR in human Sertoli cells after a 24 h CBD treatment. (B) Western blot analysis of CDKN2A/p16 was conducted in human Sertoli cells after a 24 h CBD treatment. GAPDH was used as an internal control. Representative Western blots are shown in left panel. Quantification of relative band intensity is shown in right panel. The bar graphs represent means ± SD (n=3). The intensity of each protein band was normalized to its individual GAPDH band. *, significantly different from the DMSO control. (C) The line graph shows the cumulative population doubling of human Sertoli cells treated with DMSO (0 μM) or 10 μM CBD for 12 days. (D) The bar graph shows the relative fluorescence intensity of SA-β-gal in DMSO (0 μM)- or 10 μM CBD-treated human Sertoli cells. The bar graphs represent means ± SD (n=3). *, significantly different from the DMSO control at each time point. #, significant time-related linear trend in 10 μM CBD treatment group. (E) Human Sertoli cells were stained for SA-β-gal (blue-green) after a 12-day treatment with DMSO (0 μM) or 10 μM CBD. Bright-field images are representative of three independent experiments. Scale bar is 300 μm.

We next examined the growth of DMSO (0 μM)- or 10 μM CBD-treated cells for an extended period of 12 days by calculating the cumulative population doubling. Cumulative population doubling refers to the total number of times that the cell number doubles. Calculation of cumulative population doubling is a commonly used method to measure cellular senescence in vitro, based on the fact that proliferative potential decreases during the process of cellular aging (Chen et al., 2013). On Day 12, the cumulative population doubling of the control group (0 μM, DMSO) was 14, whereas the cumulative population doubling of the CBD-treated group was −0.19 (p>0.05 compared to Day 0), suggesting that the cells of control group grew continuously while CBD-treated cells stopped proliferating starting at the time of exposure to CBD, because the cell number remained approximately the same from Day 0 to Day 12 (Figure 6C).

We then investigated the phenotypic character of cellular senescence by measuring activation of SA-β-gal. We performed flow cytometry analysis to quantify the changes of the activity of SA-β-gal for up to 12 days. As shown in Figure 6D, we observed that the activity of SA-β-gal in 10 μM CBD-treated cells increased in a time-dependent manner. From Day 6 to Day 12, the SA-β-gal activity in 10 μM CBD-treated cells was significantly higher than that of the corresponding DMSO control (Figure 6D). In addition, we conducted SA-β-gal staining on Day 12 to directly visualize the activation of SA-β-gal. We observed that nearly 100% of the cells were stained positive (blue-green) after continuous exposure to 10 μM CBD for 12 days (Figure 6E), whereas DMSO-treated cells were stained negative. These findings suggest that CBD treatment caused cellular senescence in human Sertoli cells.

Discussion

Despite the popular uses of CBD products, their safety profile is still not fully understood. Previously, we reported that CBD induced cytotoxicity in human and mouse Sertoli cells. Herein, we investigated the molecular mechanism underlying the cytotoxicity of CBD in human Sertoli cells using mRNA-seq analysis. In this study, the concentrations of CBD used were 7–10 μM, which are within an order of magnitude of the CBD plasma levels measured in humans. For example, the Cmax of CBD in plasma ranged from 0.9 to 2.3 μM in healthy volunteers consuming 1500 mg/day CBD for seven consecutive days (Taylor et al., 2018). High-fat diets and preexisting liver disease can increase the Cmax of CBD in plasma (Taylor et al., 2018). Specifically, the concentration of CBD in plasma increased 5.9-fold in those who eat high-fat meals and 1.5 to 5.2-fold in patients with hepatic impairment (Taylor et al., 2019; Taylor et al., 2018).

Among the significantly changed genes, we noticed that some genes mapped to multiple pathways and networks. For example, cell cycle-related kinases, cyclins, p21, GADD45A, and growth arrest and DNA damage inducible gamma (GADD45G) can participate in cell cycle and p53 signaling pathways; proteins encoded by DNA replication factor 1 (CDT1), cell division cycle 6 (CDC6), cell division cycle 7 (CDC7) and origin recognition complex (ORC) function in both cell cycle and DNA replication pathways; and single-stranded DNA-binding protein RPA and DNA polymerases play important roles in both DNA replication and DNA repair. To prevent redundancy, the genes that can be classified to multiple pathways were illustrated in only one pathway in our heatmaps.

E2F7, a transcriptional repressor, appears to be the direct regulator for gene expression changes in DNA replication, cell cycle progression, and DNA repair pathways (Aksoy et al., 2012; Bracken et al., 2004; Westendorp et al., 2012). Some examples of E2F7 target genes are (1) DNA replication-related: CDC6, CDT1, MCM2–7, PRIM1, POLA1, proliferating cell nuclear antigen (PCNA), replication factor C (RFC)2, RFC4, and flap structure-specific endonuclease 1 (FEN1); (2) cell cycle-related: polo like kinase 1 (PLK1), cyclin A (CCNA), cyclin B (CCNB), cyclin E (CCNE), and CDK1; (3) DNA repair-related: mutS homolog 2 (MSH2), MSH6, EXO1, RAD51, and Fanconi anemia complementation group I (FANCI). In contrast, E2F1 is a transcriptional activator that upregulates expression of E2F target genes such as CDC25A, cyclin E1 (CCNE1), and cyclin E2 (CCNE2) (Vigo et al., 1999). In addition, E2F1 is a direct target of E2F7 (Aksoy et al., 2012). In this study, we observed a downregulation of E2F1, which may result from the increase of E2F7 (Figure 2A). Additionally, E2F2 is homologous to E2F1 and therefore has a very similar function to E2F1 in regulating E2F targets (Timmers et al., 2007).

One significant finding of our study, first from transcriptomic analysis and then validated using independent methods, is that CBD treatment induced senescence. It is known that cellular senescence mostly occurs in the G1 phase of the cell cycle and that senescent cells stop synthesizing DNA and are resistant to apoptosis (Kumari and Jat, 2021). These observations are consistent with our previous findings that CBD induced G1 cell cycle arrest, inhibited DNA synthesis activity, and exhibited an absence of apoptosis in human Sertoli cells (Li et al., 2022).

It has been reported that long-lasting SASP mediates harmful effects because the chronic presence of SASP promotes local and systemic inflammation (Kumari and Jat, 2021), which can eventually result in premature aging (Chung et al., 2009; Franceschi and Campisi, 2014; Frungieri et al., 2018). An increase of IL-6 protein, which plays important roles in SASP, has been shown to induce germ cell apoptosis (Białas et al., 2009). A study reported that drug-induced inflammatory responses, such as peritubular macrophage infiltration into the testes, play a crucial role following germ cell injury and aiding in recovery of spermatogenesis (Gillette et al., 2021). On the other hand, a study reported that SASP inhibited the function of macrophage in fibroblast (Ogata et al., 2021). The interaction of SASP and immune cells in reproductive system warrants further investigation. Nevertheless, our results regarding the SASP induction, particularly, the increase of IL-6, further reinforce that CBD might have a harmful effect on the male reproductive health.

Eight groups of small chemicals or agents have the potential to induce cellular senescence and senescence-like features: they are (1) DNA replication stress inducers; (2) DNA damaging agents, such as DNA topoisomerase inhibitors and DNA cross-linkers; (3) epigenetic modifiers, such as DNA methyltransferases inhibitors; (4) telomerase inhibitors; (5) CDK inhibitors; (6) activators of p53; (7) activators of protein kinase C; and (8) reactive oxygen species inducers (Petrova et al., 2016). Using this classification, CBD appears to fit “p53 activators” because we observed the upregulation of p53 and the activation of p53 via phosphorylation of Ser 15. The activation of p53 was additionally validated by monitoring the nuclear transportation of p53 on day 3, 6, 9, and 12 after 10 μM CBD treatment using flow cytometry (MultiFlow DNA Damage Kit from Litron Laboratories). The increase of nuclear p53 implicates its activation (Supplementary Figure 1). Numerous studies have demonstrated an important role of p53 in cellular senescence. For instance, disruption of p53 function was reported to stop fibroblasts from entering senescence (Webley et al., 2000). Increased Ser 15 phosphorylation of p53 was found in replicative senescence and stress-induced senescence (Kumari and Jat, 2021; Petrova et al., 2016; Webley et al., 2000). It is interesting that Ser 15 phosphorylation of p53 was commonly seen in DNA damage response or damage-related senescence phenotypes (Kumari and Jat, 2021). CBD has been suggested to induce DNA damage in human hepatic HepG2 cells (Russo et al., 2019) and mouse sperm cells (Carvalho et al., 2022; Russo et al., 2019). However, we did not observe an increase of γ-H2AX, a hallmark of DNA damage, and we did not find DNA strand breaks with Comet assays after 3 h and 24 h 10 μM CBD treatments in human Sertoli cells (data not shown). This indicates that CBD did not cause DNA damage in human Sertoli cells, even though p53 was activated. It is not clear why CBD-induced DNA damage was not observed in our study, and this inconsistency warrants further investigation; it may be due to distinct responses in different cell types and the different concentrations used.

In summary, as depicted in Figure 7, we have demonstrated that CBD activates p53 and upregulates p16 in human Sertoli cells. The activation of p53 can upregulate p21signaling pathways. The upregulation of p21 and p16 can activate their transcriptional repression activities, such as DNA replication, cell cycle, and DNA repair, which eventually leads to cellular senescence. Our results demonstrate that multiple signaling pathways and multiple molecular events contribute to CBD-induced senescence and cytotoxicity in human Sertoli cells. Based on the totality of evidence, one could speculate that CBD-induced senescence and cytotoxicity in Sertoli cells might lead to premature functional ageing of the testes with potential adverse reproductive consequences.

Figure 7. A scheme for a proposed mechanism underlying CBD-induced cytotoxicity in human Sertoli cells.

Figure 7.

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CBD treatment activates p53 in human Sertoli cells, increases downstream p21, which are important in inhibiting DNA replication-, DNA repair-, and cell cycle-related gene transcription. The increase of p16 can contribute to prolonged cell cycle arrest in human Sertoli cells leads to cellular senescence.

Supplementary Material

Supplementary Table

Acknowledgments:

This work was supported by U.S. Food and Drug Administration’s intramural grant program.

Footnotes

Compliance with ethical standards

Conflict of interest: The authors declare no conflict of interest.

Disclaimer: This article reflects the views of the authors and does not necessarily reflect those of the U.S. Food and Drug Administration. Any mention of commercial products is for clarification only and is not intended as approval, endorsement, or recommendation.

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