Cannabidiol Inhibited Reproductive Ability of Zebrafish (Danio Rerio) with Gender-Dependent Manner

doi:10.21203/rs.3.rs-1990207/v1

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Cannabidiol Inhibited Reproductive Ability of Zebrafish (Danio Rerio) with Gender-Dependent Manner

https://doi.org/10.21203/rs.3.rs-1990207/v1

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Cannabidiol (CBD), a medically active component of hemp, is a popular ingredient in healthcare and personal-care products. The increasing demand for CBD and the legalization of hemp growth may promote chronic exposure of non-target organisms and animals to CBD. In the present study, the reproductive toxicity of CBD was investigated using zebrafish as a model organism. With CBD treatment, parent female zebrafish spawned less with higher natural mortality and malformation rates, showed a decreased gonadosomatic index with an increased percentage of pre-mature oocytes and sperm and had an increased hepatosomatic index. The value of estrogen/testosterone (E2/T) decreased in female zebrafish and increased in male zebrafish. Vitellogenin content was decreased in both female and male zebrafish. Reproductive-related genes were downregulated in the zebrafish brain and liver, whereas esr-α, esr-β, and ar were downregulated in ovaries and upregulated in testicles. Sex hormone synthesis genes were downregulated in ovaries and upregulated in testicles, except for cyp11a, in contrast to the other genes. Apoptosis-related genes were upregulated in the zebrafish brain, gonad, and liver. These results show that CBD might damage the hypothalamus-pituitary-gonad-liver axis function by inducing an apoptotic response, further inhibiting zebrafish reproductive ability in a sex-dependent manner.

Cannabidiol

Zebrafish

Reproductive Toxicity

Gonad

Sex-Dependent Manner

Cannabidiol (CBD), a phyto-cannabinoid isolated from hemp (Cannabis sativa L.), has been proposed as the most promising medical component in hemp because of its anti-inflammatory and antioxidant properties, especially since it demonstrates non-addictive activity (Gülck and Møller, 2020, Sainz-Cort et al., 2021). Recently, hemp growth has been legalized in more than 50 countries, which has promoted CBD as a functional component in foods, drinks, and cosmetics (Berger et al., 2021). There is also an FDA (US Food and Drug Administration)-approved pharmaceutical, Epidiolex, whose active ingredient is CBD that targets seizures with Lennox-Gastaut and Dravet syndrome (USFDA, 2018). Increasing consumption of CBD leads to the release of CBD into the environment, including factory emissions and personal excretion (How and Gamal El-Din, 2021). Mastroianni et al. (2013) reported tetrahydrocannabinol, CBD, and cannabinoid (CBN) in sewage sludge in Mediterranean watersheds in Spain with concentrations of 29.9‒579.0, 32.3‒479.0, and 26.7‒188.0 ng/g, respectively. In recent years, the legalization of hemp growth may have promoted CBD and other cannabinoids contamination in water, which may lead to chronic exposure of aquatic organisms and animals, with unexpected results.

CBD at 150 µg/L did not induce any developmental alterations in zebrafish embryos, whereas CBD beyond 300 µg/L significantly reduced the survival and hatching rate of zebrafish embryos, decreased heart rates and length of trunks, and increased axial malformations (Carty et al., 2018, Valim Brigante et al., 2018), suggesting that CBD exerted a concentration-dependent effect on zebrafish embryos. CBD also has a negative effect on zebrafish mobility by decreasing swimming speed and distance (Jensen et al., 2018). Ahmed et al. (2018) found that CBD inhibits the development of secondary motor neurones and decreases the quantity of nicotinic acetylcholine receptors in zebrafish embryos, which might be the reason for the decreased mobility and slow response to exogenous stimuli. CBD also induced reproductive toxicity in zebrafish. Exposure of larvae to CBD reduced sperm production in male zebrafish and egg production in female zebrafish (Pandelides et al., 2020a, Pandelides et al., 2020b). However, there has been no further investigation into the mechanism of reproductive toxicity of CBD in zebrafish.

A study on Swiss mice showed that CBD negatively affected the reproductive system with decreased frequency of spermatogenesis in stages VII‒VIII and XII, and increased the frequency of sperm with head abnormalities and cytoplasmic droplets in the medial region of the flagellum (Carvalho et al., 2018). A study on human cell lines found that CBD inhibited endometrial stromal cell differentiation into secretory cells, an effect that is detrimental to placental formation and early embryonic growth (Neradugomma et al., 2019). Cell experiments have also shown that CBD increases the permeability of the placenta to xenobiotics, which may be harmful to the developing foetus (Feinshtein et al., 2013). These studies suggest that CBD functions in multiple ways to induce reproductive toxicity in vertebrates.

There is no direct clinical evidence that CBD has a deleterious effect on foetal development. Due to the inexpensive cost, claimed therapeutic advantages, and indeterminate adverse effects of CBD or cannabis, CBD has been commonly used by pregnant women to address nausea, sleeplessness, anxiety, and chronic pain associated with pregnancy (Bayrampour et al., 2019). Nonetheless, based on studies in zebrafish and mice, pregnant women and their foetuses may experience unwanted side effects if they consistently consume CBD or cannabis. Thus, pregnant and childbearing women should be extremely cautious when using CBD.

In this study, the reproductive toxicity of CBD on adult zebrafish was investigated to determine how CBD alters the reproductive system of adult zebrafish and the effect of CBD on the offspring. The evaluation of the reproductive capacity of adult zebrafish was based on the quantity and mortality of embryos. The malformation rate and body length were used to assess the impact of CBD exposure on the progeny. Gonad histology, sex hormone and vitellogenin levels, and mRNA levels of reproduction-related genes were determined to further illustrate how CBD affects the reproductive system of adult zebrafish.

2.1 Chemicals

CBD (99% purity) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd., China. CBD was dissolved in dimethyl sulfoxide (DMSO) as a stock solution (10 g/L) and stored at 4°C. Acetonitrile and formic acid were LC-MS-grade (Thermo Fisher Scientific, USA). Pure water was obtained from a Milli-Q ultrapure water system with a resistivity of 18.3 MΩ*cm (25°C). Other chemicals were analytical-grade and purchased from local chemical vendors.

2.2 Zebrafish maintenance

Embryos of zebrafish (Danio rerio, AB type) were acquired from the China Zebrafish Resource Center (CZRC, Wuhan, China) and maintained following the standard feeding protocol (Supplementary I, from CZRC) until 5 months of age for reproductive experiments. Zebrafish over 1 month were kept in circulating and fully aerated water with a temperature of 27 ± 1°C, salinity of 0.35 ± 0.1%, conductivity of 500 ± 100 µS/cm, pH between 7.0 and 8.0, and photoperiod of 14 h light:10 h dark. Zebrafish were fed twice a day with the proper amount of newly hatched brine shrimp, which was ate up within two minutes.

Adult zebrafish were domesticated using the semi-static method with circulating water (changed by approximately 2/3 every day) for 14 days to adapt to the feeding method and environment. Throughout the 14-day period, the zebrafish spawned every seven days in the morning following light stimulation. Zebrafishes of comparable size and fecundity were selected for acute toxicity and short-term reproduction tests.

The study was approved by the ethics committee of the Institute of Food Science and Technology for research on laboratory animals and was conducted under the guidelines for the care of laboratory animals of the Chinese Academy of Agricultural Sciences (CAAS).

2.3 Acute toxicity test

Adult zebrafish were exposed to CBD (500, 600, 700, 800, and 900 µg/L) for 96 h using the semi-static method. Water containing DMSO (0.01%) was used as a control (the same as follows, DMSO water treatment did not cause zebrafish death in the pretest). Dead fish were recorded and collected daily before the water change. This test was repeated three times with three replicates for each treatment, and ten fish for each replication.

2.4 Short-term reproduction test

Based on the results of the acute toxicity test and the CBD concentration detected in sewage sludge, adult zebrafish were exposed to two sublethal concentrations of CBD for 21 days using the semi-static method. Six female or male zebrafish were in a 2 L tank for replication. Three replicates were used for one treatment. On days 7, 14, and 21, one female and one male zebrafish were placed in a breeding box without CBD for mating at 20:00. The spawn was stimulated once the light was turned on in the next morning for 1 h. Thereafter, adult zebrafish were returned to the exposure solution. The embryos were recorded and moved to Holt buffer (1 L distilled water containing 3.5 g NaCl, 0.05 g KCl, 0.025 g NaHCO₂, and 0.1 g CaCl₂, changed every day) for 4 days for body length, mortality, and malformation recording.

2.5 Zebrafish dissection

At the end of 21 days of exposure, all zebrafish were anaesthetised and sacrificed with 0.2 mg/L MS222. Surface water was removed with paper, weighed individually, and dissected for the brain, liver, ovary, testicle, and tail (from gonopore to the end without caudal fin). For each sex from one replicate in each treatment, three tails were weighed and frozen for sex hormone and vitellogenin (Vtg) determination: one ovary/testicle was fixed in 4% paraformaldehyde at 4°C for paraffin sectioning, two brains/livers/ovaries/testicles were collected for CBD detection, and three brains/livers/ovaries/testicles were collected for weighing and then frozen in liquid nitrogen for RNA extraction. Gonad and liver weights were used to calculate the gonadosomatic index (GSI) and hepatosomatic index (HSI).

2.6 Histological observation of ovary and testicle

The fixed gonads were dehydrated using a series of concentrations of ethanol (80%, 90%, 95%, and 100%) and xylene (100%) and embedded in paraffin. Sections of approximately 5 µm were cut and stained with haematoxylin and eosin (Cao et al., 2016), then observed under a microscope. The number of different types of oocytes was counted under a 40× microscope, and the area of the sperm was quantified using ImageJ 1.53k (National Institutes of Health, USA) with pictures taken under a 200× microscope.

2.7 RNA extraction, cDNA synthesis, and qRT-PCR

RNA from the brain, ovary, testicle, and liver of zebrafish was extracted with kits from TransGen Biotech (Beijing, China) and detected using a nano-photometer (NP80). cDNA was synthesised (TransGen Biotech) for quantitative real-time polymerase chain reaction (qRT-PCR) (ABI Q7 Flex 384 well Real-Time PCR System). The mRNA levels of genes were calculated using the 2^−ΔΔCT method. Primers for the reference and target genes are shown in Supplementary Table S1.

2.8 CBD detection in zebrafish organs and exposure solution

Zebrafish organs were homogenised with 1 mL trichloromethane, vortexed vigorously for 2 min, sonicated for 10 min, and centrifuged at 8000 × g for 10 min at room temperature. The supernatant was transferred to a new tube, dried under nitrogen, redissolved with 0.2 mL acetonitrile, and filtered through a 0.22-µm membrane.

Because the exposure solution was changed every day during the short-term reproduction test, it was examined for CBD concentrations before and after changing. Two millilitres of the exposure solution were added with 1 mL trichloromethane and vigorously shaken for 2 min. The organic phase was transferred to a new tube, dried under nitrogen, redissolved in 1 mL acetonitrile, and filtered through a 0.22-µm membrane.

CBD detection was conducted using ultra-performance convergence chromatography/Xevo triple quadrupole mass spectrometry (UPLC-Xevo TQXS System, Waters) with an ACQUITY UPLC BEH C18 column (2.1 × 50 mm, 1.7 µm). The mobile phases were pure water with 0.1% formic acid and acetonitrile with 0.1% formic acid (Supplementary Table S2). CBD was detected with multiple reaction monitoring in positive ion (ES⁺) mode with a cone voltage of 40 V, collision voltage of 25 eV, dwell time of 0.025 s, parent ion of 315.1 m/z, and daughter ion of 193.2 and 135.1 m/z (Zhang et al., 2015a).

2.9 Data analysis

OriginPro 2021 software (OriginLab Corporation) was used in this study. Acute toxicity in adult zebrafish was analysed using probit regression analysis. The adult productivity, offspring malformation rate, and mortality of control and CBD treatments were compared by one-way ANOVA with a Duncan post-hoc test and a significance level of 0.05. The same method was used to compare mRNA levels between the control and CBD treatments.

3.1 Chemical analysis

3.1.1 Method validation

The added levels of CBD in the zebrafish samples were 0.01, 0.05, 0.1, and 0.5 µg/g. The average spiked recovery of CBD in fish samples was 81.71 ± 4.19% (Supplementary Table S3). The added concentrations in the water samples were 1, 10, and 100 µg/L. The average spiked recovery of CBD in the water samples was 80.08 ± 4.92%. The limit of detection of the methods in fish and water samples was 0.025 µg/kg and 6.0 µg/L, and the limit of quantification was 0.075 µg/kg and 18.0 µg/L, respectively. The CBD spectrum is shown in Supplementary Figure S1.

3.1.2 CBD analysis in exposure solution

The results of the chemical analysis of CBD are shown in Supplementary Table S4. The results indicated that the deviations of CBD concentrations at 0 and 1 d after the exposure solution change were maintained within ± 20%. Thus, in short-term exposure tests, nominal concentrations can represent the actual concentrations of CBD (OECD, 2012).

3.2 Acute toxicity of CBD on adult zebrafish

CBD showed lethal toxicity in adult zebrafish. A regression equation was constructed with the probability of mortality (y) and log (concentration) (x) as follows: y = 10.09x − 28.39 (R² = 0.9472). The 96 h-LC₅₀ of CBD on adult zebrafish was 649.4 µg/L (614.4–682.8 µg/L with 95% confidence limits). In the following tests, 200 and 300 µg/L were selected as sub-lethal concentrations for the short-term exposure test.

3.3 CBD decreased the reproductive ability of adult zebrafish

First, CBD inhibited the reproductive ability of zebrafish. On one hand, the spawned eggs declined about 15%, 20%, and 20% in 200 µg/L treatment, and about 20%, 27%, and 36% in 300 µg/L treatment in weeks 4, 5, and 6, respectively (p < 0.05, Fig. 1a). Additionally, reproductive behaviour was significantly inhibited in female zebrafish by CBD (p < 0.05, Fig. 1b). The inhibition on fecundity and reproductive behaviour followed concentration dependent manner, which was in agreement with a previous study in which zebrafish exposed to CBD showed decreased oviposition (Pandelides et al., 2020a). Second, CBD exposure had serious side effects on the offspring, such as significantly increased natural mortality and malformation rates (p < 0.05, Fig. 1c and 1d). Malformation in the offspring was observed as pericardial oedema, enlarged abdomen, decreased melanin, maldeveloped eyes, and curved body (Fig. 1e). Additionally, the body length of normal-shaped offspring in the CBD treatment group was significantly shorter than that in the control group (p < 0.05, Fig. 1f and 1g).

3.4 Accumulation of CBD in zebrafish organs

CBD has been detected in reproductive organs of zebrafish. As shown in Table 1, in the same sex, the content of CBD in the brain and gonads increased with CBD exposure. In the same treatment and same organ, the CBD content was higher in male zebrafish than in female zebrafish. However, the CBD content in the liver did not follow these rules. CBD content increased with exposure concentration in the female liver but decreased in the male liver. CBD accumulated similarly in the female and male livers in the 200 µg/L treatment and accumulated more in the female liver than in the male liver in the 300 µg/L treatment.

Table 1

Content of CBD in zebrafish organs after 21 days exposure
Organs	Treatment	Female (µg/g)	Male (µg/g)
Brain	Control	ND Aa	ND Aa
	200 µg/L	0.2592 + 0.0174 Bb	0.4573 + 0.0377 Ab
	300 µg/L	0.4552 + 0.0414 Bc	0.6567 + 0.0351 Ac
Gonad	Control	ND Aa	ND Aa
	200 µg/L	0.7549 + 0.0644 Bb	2.4095 + 0.1285 Ab
	300 µg/L	1.3156 + 0.0182 Bc	2.7217 + 0.1704 Ac
Liver	Control	ND Aa	ND Aa
	200 µg/L	0.4254 + 0.0237 Ab	0.4343 + 0.0503 Ac
	300 µg/L	0.5529 + 0.0893 Ac	0.3442 + 0.0396 Bb
Note: Two organs from female or male zebrafish were pooled as one replicate. Data were calculated from three replicates. ND means no detection. Lowercase letters indicate significant differences between treatments from control and CBD treatments in the same sex and same organs compared by ANOVA, while uppercase letters indicate significant differences between females and males in the same organ and same treatment compared by T-test (p < 0.05).

3.5 Effect of CBD on gonad and liver of zebrafish

After 21 d of exposure, CBD significantly inhibited gonad development and induced hepatomegaly in both female and male zebrafish. The GSI decreased and HSI increased in both female and male zebrafish in the CBD exposure treatments compared with the control (Fig. 2). Gonad and liver directly regulate the reproductive behaviour in fish. The alternation of GSI and HSI reflect the condition of fish, which are in relation to the environmental change and toxin exposure (Singh et al., 2015). GSI is used to estimate maturity staging of fish, which indicates immature or damaged gonad in fish with decreased value of GSI. The alternation of GSI in this study suggested negative effect on zebrafish gonad by CBD exposure. HSI is to evaluate the condition of liver, which indicates liver pathology with abnormal value. Increased value of HSI suggests hepatomegaly, which will finally affect the balance of Vtg in zebrafish (Baumann et al., 2020).

3.6 Effect of CBD on contents of hormone and Vtg

After 21 days of exposure, the Vtg content decreased in both female and male zebrafish exposed to CBD in a concentration-dependent manner (Table 2). In female zebrafish, the content of E2 decreased and the content of T increased; the opposite occurred in male zebrafish. The value of E2/T decreased in female zebrafish but increased in male zebrafish in the CBD treatments compared to the control (Table 2).

Table 2

Content of Vtg, E2, and T in adult zebrafish tail
Sex	Treatment	Vtg (µg/g)	E2 (ng/g)	T (ng/g)	E2/T
Female	Control	768.08 ± 9.87 a	397 ± 11.05 a	183.94 ± 7.33 b	2.16 ± 0.03 a
	200 µg/L	516.98 ± 10.05 b	275.64 ± 10.69 b	212.85 ± 5.73 a	1.29 ± 0.05 b
	300 µg/L	389.18 ± 9.10 c	236.61 ± 7.38 c	222.26 ± 4.90 a	1.06 ± 0.02 b
Male	Control	686.89 ± 8.89 A	207.79 ± 7.75 C	299.36 ± 6.55 A	0.69 ± 0.03 C
	200 µg/L	618.09 ± 13.84 B	234.60 ± 5.60 B	266.23 ± 6.34 B	0.90 ± 0.03 B
	300 µg/L	525.52 ± 14.18 C	257.99 ± 6.66 A	253.27 ± 4.93 B	1.01 ± 0.01 A
Note: Data are shown as the MEAN ± SD from three replicates (tails from three zebrafish were pooled as one replicate). Different lowercase and uppercase letters indicate significant differences between the two treatments in female and male zebrafish, respectively (p < 0.05).

In zebrafish, E2 and T cooperate to regulate sexual and reproductive behaviours. Both female zebrafish exposed to 11-ketotestosterone (lower value of E2/T) and male zebrafish exposed to 17α-ethynylestradiol (higher value of E2/T) exhibited reduced mating and spawning behaviour, implying masculinisation of female sex hormones in male zebrafish and feminisation of male sex hormones in female zebrafish (Pradhan and Olsson, 2015). A previous study showed that damaged reproductive function in zebrafish is correlated with lower E2/T values in female zebrafish and higher E2/T values in male zebrafish (Cao et al., 2016). Therefore, the variation in E2 and T content has become a sign for evaluating the reproductive ability of zebrafish. In this study, the decreased E2/T value in female zebrafish and increased E2/T value in male zebrafish suggested damaged gonad function in zebrafish.

Female sex hormones are beneficial for the synthesis of Vtg, a nutritional source for oocyte and sperm development. External female sex hormones (E2, 17α-ethinylestradiol and 17 β-estradiol) were found to promote the synthesis of Vtg in zebrafish (Rose et al., 2002). Flouriot et al. (1996) reported that E2 stimulates vitellogenin synthesis in the liver. In this study, a correlation was observed between decreased E2 levels and Vtg content in female zebrafish. However, E2 content increased, while Vtg content decreased in male zebrafish, demonstrating that factors other than E2 content may alter the synthesis of Vtg. However, the relationship between T and Vtg is not straightforward. T is the precursor of E2, whose conversion is assumed to regulate sexual behaviour (Pradhan and Olsson, 2015). 3.7 Effect of CBD on histological structure of zebrafish gonads

CBD negatively affects the development of the ovaries and testicles of zebrafish. Figure 3a-c and Fig. 4a-c show the histological structures of the ovaries and testicles, respectively. In ovaries from the CBD treatment group, the percentage of late vitellogenic oocytes was significantly decreased, whereas the percentage of perinucleolar oocytes was significantly increased compared to the control (Fig. 3d, p < 0.05). In testicles from CBD treatments, the percentage area of spermatids and spermatozoa was lower than that in the control (Fig. 4d, p < 0.05).

3.8 Effect of CBD on reproduction-related gene expression

In the zebrafish brain, the expression of reproductive-related genes, gnrh, fsh-β, lh-β, and lh-γ, was significantly downregulated in the CBD treatment group compared to that in the control (Fig. 5a, p < 0.05). The mRNA levels of gnrh, fsh-β, lh-β, and lh-γ in the 200 µg/L and 300 µg/L treatments were about 5%, 27%, 27%, 12%, and 34%, 54%, 6%, and 61% of the control in the female brain, and were approximately 0.1%, 12%, 3%, 37%, and 0.4%, 13%, 1%, and 55% of the control in the male brain, respectively.

In the zebrafish, multiple hormones affect the reproductive behaviour of zebrafish in a systemic and sequential manner. In the brain, the hypothalamus secretes gonadotropin-releasing hormone (GnRH), which stimulates the synthesis and release of gonadotropins, including the glycoprotein molecules luteinizing hormone (LH) and follicle-stimulating hormone (FSH) (Gore, 2002). Subsequently, LH and FSH are transported into the gonads to promote the synthesis and secretion of sex steroid hormones such as oestrogen (E2) and testosterone (T) (Gore, 2002). Finally, E2 acts on the liver to encourage the production and secretion of Vtg, which then operates in the gonad to nourish the developing oocyte (Gore, 2002). Studies have shown that GnRH depletion is positively correlated with a reduction in GSI and gonadotropin production, further affecting oogenesis, spawning success, fecundity, and male courting behaviour (Marvel et al., 2021). LH and FSH also positively affect the development of zebrafish gonads. However, they work in different ways in the hypothalamus-pituitary-gonad-liver axis (HPGL axis). FSH deficiency delays the development of ovaries and testicles in zebrafish, but has no effect on the spawning process in female zebrafish, whereas LH deficiency did not affect the growth of gonads, but made the female zebrafish unable to spawn (Zhang et al., 2015b). In this study, CBD treatment reduced the number of spawned eggs in zebrafish, which was not only due to a decrease in egg production per female, but also a reduction in the number of spawning zebrafish (Fig. 1a and 1b). Considering the decreased mRNA levels of gnrh, lh-α, lh-β, and fsh in CBD treatments, CBD exposure was concluded to have a negative effect upstream of the HPGL axis.

FSH and LH are transported by the blood to the gonads, where they stimulate the production of E2 and T. In the ovary, all genes related to E2 and T synthesis were downregulated, except for cyp11a, which was upregulated. In the testicles, all genes were upregulated, except cyp11a, which was downregulated. In ovaries, esr-α, esr-β, and ar were downregulated following CBD treatment. In testicles, esr-α, esr-β, and ar were upregulated by CBD treatment (Fig. 5b, p < 0.05). The mRNA variations of the E2 receptor (esr1 and esr2, decreased in the ovary and decreased in the testicle) and T receptor (ar, decreased in ovary and increased in testicle) were corroborated by the variation of the two sex hormones, since there was compensation of androgen receptor for decrease of T in zebrafish, while no such phenomenon was observed in relation to E2 receptor and E2 in zebrafish (Smolinsky et al., 2010).

The steroid hormones E2 and T are sequentially produced by the cytochrome P450 superfamily of enzymes. The cyp11a gene encodes a cholesterol side-chain cleavage enzyme that converts cholesterol to pregnenolone, which is the initial and rate-limiting step in hormone synthesis (Miller and Gucev, 2014). In this study, cyp11a was upregulated in ovaries, but downregulated in testicles after CBD treatment. Subsequently, the downstream genes were downregulated in the ovary but upregulated in the testicle (Fig. 6). CBD behaved differently in male and female zebrafish, as seen by these results. However, the opposite behaviour of cyp11a and downstream genes in E2 and T biosynthesis contributed to the disrupted function of gonads in zebrafish treated with CBD.

In both female and male livers, the mRNA levels of vtg1 and vtg2 were decreased by CBD treatment, by less than 1% compared with the control (Fig. 5c, p < 0.05). In all oviparous and ovoviviparous vertebrates, oocyte development is directly governed by Vtg, the precursor of yolk protein, which is secreted into the plasma by the liver and absorbed and cleaved by growing oocytes to provide nutrition for oocyte development (Sullivan and Yilmaz, 2018). Wu et al. (2021) found that zebrafish containing a lower Vtg content in plasma gained an increased number of early vitellogenic oocytes, indicating delayed development of follicles, which was in accordance with the performance of Vtg content and histological structure of oocytes in CBD treatments in this study. Apart from the delayed development of oocytes, there were some atretic oocytes in the CBD treatment group (Fig. 3b and 3c). Atretic oocytes commonly occur in zebrafish that are aged, dystrophic, or under environmental stress (Brooks et al., 1997). Liu et al. (2017) found that a decreased level of Vtg induced the occurrence of atretic oocytes in female zebrafish. In this study, CBD exposure might be responsible for the occurrence of atretic oocytes, since all tested zebrafish were maintained in good conditions, and there were no atretic oocytes in the ovaries of the control zebrafish. Vtg was also correlated with malformations and mortality in zebrafish offspring. Peng et al. (2019) found that lower levels of Vtg were detected in phenanthrene-treated parent zebrafish, whose offspring showed decreased hatching rates and increased malformation and mortality rates. In conclusion, CBD affected the HPGL axis of parent zebrafish, resulting in the delayed and aberrant development of oocytes and sperm.

3.9 Effect of CBD on apoptosis-related gene expression

CBD induces apoptosis in zebrafish reproductive-related organs. The mRNA levels of casp8, casp9, casp3, and cyt were higher than those in the control in female and male brains (Fig. 5d). Similar results were observed in the gonads and the liver (Fig. 5e and 5f). Among the four genes, the mRNA level of cyt in the male reproductive organs was higher than that in the female reproductive organs.

Apoptosis is a cell-autonomous process that prevents serious tissue reactions to stimuli (Fink and Cookson, 2005). Production of cytochrome c is an initiation response to apoptotic stimuli and a subsequent stimulation of the caspase cascade (Ow et al., 2008). In the CBD treatments, cyt (coding for cytochrome c) was significantly upregulated in three organs of both female and male zebrafish, indicating the possibility of CBD-induced apoptosis in zebrafish reproductive-related organs. The results showed that the mRNA levels of cyt were higher in male organs than in female organs, suggesting that CBD induced more serious apoptosis in male zebrafish, which was in accordance with CBD accumulation in zebrafish organs (Table 1). In the caspase cascade, caspase-9, an initiator of intrinsic apoptosis, is required for reactive oxygen species (ROS) production and caspase-3 activation, whereas caspase-3 is required for the suppression of ROS production and execution of apoptosis (Brentnall et al., 2013). Thus, the two proteins were typically upregulated or downregulated simultaneously, which was consistent with the outcome of CBD treatment. The accumulation of caspase-9 in the nucleus is a sign of apoptosis in the hippocampus and a marker of brain injury (Krajewski et al., 1999). Caspase-8 is an initiator of extrinsic apoptosis and a molecular switch for necroptosis, and its activation mediates death receptor-dependent apoptosis (Newton et al., 2019). In this study, caspase-3, caspase-9, and caspase-8 were all upregulated in three organs of female and male zebrafish, indicating that CBD triggered cell apoptosis in these organs, potentially resulting in tissue damage and dysfunction of the HPGL axis. The probable tissue damage and dysfunction in the liver might also explain why increased E2 content led to decreased Vtg content instead of increased Vtg content in male zebrafish.

In conclusion, CBD reduced the number of spawned eggs and increased the mortality and malformation rates of offspring. Furthermore, CBD reduced GSI, increased HSI, interfered with E2, T, and Vtg balance, and induced abnormal gonad development. Genes related to apoptosis (cyt, casp3, casp8 and casp9) were upregulated in the brain, liver, and gonads, indicating that CBD might damage the function of the HPGL axis by inducing apoptosis in these organs. Genes related to hormone synthesis (cyp11a and downstream genes) were differentially expressed in female and male zebrafish, indicating that CBD functions in zebrafish in a sex-dependent manner. These results suggest that CBD negatively affected the reproductive ability of zebrafish.

CBD, cannabidiol; intake; E2, estrogen; T, testosterone; HPGL axis, hypothalamus-pituitary-gonad-liver axis; THC, tetrahydrocannabinol; CBN, cannabinoid; OR, odd ratio; DMSO, dimethylsulfoxide; Vtg, vitellogenin; GSI, gonadosomatic index; HSI, hepatosomatic index; LOD, limit of detection; LOQ, limit of quantity; LC₅₀, median lethal concentration; GnRH, gonadotropin-releasing hormone; LH, luteinizing hormone; FSH, follicle-stimulating hormone; HPGL axis, hypothalamus-pituitary-gonad-liver axis. ROS, reactive oxygen species.

Acknowledgements We would like to thank Prof. Demetrio Raldua Perez from the Institute for Environmental Assessment and Water Research (IDAEA-CSIC) for his advice during manuscript writing. We would like to thank Editage (www.editage.cn) for English language editing.

Author contribution LI Lin: Methodology, Formal analysis, Writing-original draft. FAN Bei: Conceptualization. KONG Zhiqiang: Formal analysis, Writing - review & editing. ZHANG Yifan: Methodology. ZHAO MengYing: Methodology. Jesus Simal-Gandara: Formal analysis. Fengzhong Wang: Funding acquisition, Supervision. Minmin LI: Project administration, Conceptualization.

Funding This study was supported by the National Key Research and Development Program of China [grant number 2019YFC1604504], National Natural Science Foundation of China [grant number 31872006], and Beijing Nova Program of Science and Technology [grant number Z191100001119121].

Conflicts of interest All authors declare that they have no conflict of interest.

Ahmed KT, Amin MR, Shah P et al (2018) Motor neuron development in zebrafish is altered by brief (5-hr) exposures to THC (∆9-tetrahydrocannabinol) or CBD (cannabidiol) during gastrulation. Sci. Rep. 8, 10518. http://10.1038/s41598-018-28689-z
Baumann L, Holbech H, Schmidt-Posthaus H et al (2020) Does hepatotoxicity interfere with endocrine activity in zebrafish (Danio rerio)? Chemosphere 238, 124589. https://doi.org/10.1016/j.chemosphere.2019.124589
Bayrampour H, Zahradnik M, Lisonkova S et al (2019) Women's perspectives about cannabis use during pregnancy and the postpartum period: An integrative review. Prev. Med. 119, 17-23. http://10.1016/j.ypmed.2018.12.002
Berger BA, Stolz U, Colvin J et al (2021) Epidemiology of cannabidiol related cases reported in the National Poison Data System – 2019-2020. Am. J. Emerg. Med. 48, 218-223. https://doi.org/10.1016/j.ajem.2021.04.065
Brentnall M, Rodriguez-Menocal L, De Guevara RL et al (2013) Caspase-9, caspase-3 and caspase-7 have distinct roles during intrinsic apoptosis. BMC Cell Biol. 14, 32-32. http://10.1186/1471-2121-14-32
Brooks S, Tyler CR, Sumpter JP (1997) Egg quality in fish: What makes a good egg? Rev. Fish Biol. Fish. 7, 387-416. http://10.1023/A:1018400130692
Cao F, Zhu L, Li H et al (2016) Reproductive toxicity of azoxystrobin to adult zebrafish (Danio rerio). Environ. Pollut. 219, 1109-1121. http://10.1016/j.envpol.2016.09.015
Carty DR, Thornton C, Gledhill JH et al (2018) Developmental Effects of Cannabidiol and Δ9-Tetrahydrocannabinol in Zebrafish. Toxicol. Sci. 162, 137-145. http://10.1093/toxsci/kfx232
Carvalho RK, Santos ML, Souza MR et al (2018) Chronic exposure to cannabidiol induces reproductive toxicity in male Swiss mice. J. Appl. Toxicol. 38, 1215-1223. htpp://10.1002/jat.3631
Feinshtein V, Erez O, Ben-Zvi Z et al (2013) Cannabidiol enhances xenobiotic permeability through the human placental barrier by direct inhibition of breast cancer resistance protein: An ex vivo study. Am. J. Obstet. Gynecol. 209, 573.e571-573.e515. http://10.1016/j.ajog.2013.08.005
Fink SL, Cookson BT (2005) Apoptosis, pyroptosis, and necrosis: mechanistic description of dead and dying eukaryotic cells. Infect. Immun. 73, 1907-1916. http://10.1128/IAI.73.4.1907-1916.2005
Flouriot G, Pakdel F, Valotaire Y (1996) Transcriptional and post-transcriptional regulation of rainbow trout estrogen receptor and vitellogenin gene expression. Mol. Cell. Endocrinol. 124, 173-183. http://10.1016/s0303-7207(96)03960-3
Gore AC (2002) GnRH: The master molecule of reproduction. Springer, Boston, MA.
Gülck T, Møller BL (2020) Phytocannabinoids: Origins and biosynthesis. Trends Plant Sci. 25, 985-1004. https://doi.org/10.1016/j.tplants.2020.05.005
How ZT, Gamal El-Din M (2021) A critical review on the detection, occurrence, fate, toxicity, and removal of cannabinoids in the water system and the environment. Environ. Pollut. 268, 115642. https://doi.org/10.1016/j.envpol.2020.115642
Jensen HM, Korbut R, Kania PW et al (2018) Cannabidiol effects on behaviour and immune gene expression in zebrafish (Danio rerio). PLoS One 13, e0200016. htpp://10.1371/journal.pone.0200016
Krajewski S, Krajewska M, Ellerby LM et al (1999) Release of caspase-9 from mitochondria during neuronal apoptosis and cerebral ischemia. Proc. Natl. Acad. Sci. U. S. A. 96, 5752-5757. http://10.1073/pnas.96.10.5752
Liu T, Liu S, Ma L et al (2017) Oogenesis, vitellogenin-mediated ovarian degeneration and immune response in the annual fish Nothobranchius guentheri. Fish Shellfish Immunol. 66, 86-92. https://doi.org/10.1016/j.fsi.2017.05.015
Marvel M, Levavi-Sivan B, Wong TT et al (2021) Gnrh2 maintains reproduction in fasting zebrafish through dynamic neuronal projection changes and regulation of gonadotropin synthesis, oogenesis, and reproductive behaviors. Sci. Rep. 11, 6657. http://10.1038/s41598-021-86018-3
Mastroianni N, Postigo C, de Alda ML et al (2013) Illicit and abused drugs in sewage sludge: Method optimization and occurrence. J. Chromatogr. 1322, 29-37. https://doi.org/10.1016/j.chroma.2013.10.078
Miller WL, Gucev ZS (2014) Chapter 31 - Disorders in the initial steps in steroidogenesis. In New, M. I., Lekarev, O., Parsa, A. et al (Eds), Genetic Steroid Disorders. San Diego, Academic Press: 145-164.
Neradugomma NK, Drafton K, Mor GG et al (2019) Marijuana-derived cannabinoids inhibit uterine endometrial stromal cell decidualization and compromise trophoblast-endometrium cross-talk. Reprod. Toxicol. 87, 100-107. htpp://10.1016/j.reprotox.2019.05.064
Newton K, Wickliffe KE, Maltzman A et al (2019) Activity of caspase-8 determines plasticity between cell death pathways. Nature 575, 679-682. http://10.1038/s41586-019-1752-8
OECD (2012) Section 2: Effects on biotic systems No. 229: Fish short term reproduction assay.(Eds), OECD Guidelines for the testing of Chemicals. Paris, Organization for Economic Cooperation and Develepment.
Ow Y-LP, Green DR, Hao Z et al (2008) Cytochrome c: Functions beyond respiration. Nat. Rev. Mol. Cell Biol. 9, 532-542. http://10.1038/nrm2434
Pandelides Z, Thornton C, Faruque AS et al (2020a) Developmental exposure to cannabidiol (CBD) alters longevity and health span of zebrafish (Danio rerio). GeroScience 42, 785-800. http://10.1007/s11357-020-00182-4
Pandelides Z, Thornton C, Lovitt KG et al (2020b) Developmental exposure to Δ(9)-tetrahydrocannabinol (THC) causes biphasic effects on longevity, inflammation, and reproduction in aged zebrafish (Danio rerio). GeroScience 42, 923-936. htpp://10.1007/s11357-020-00175-3
Peng X, Sun X, Yu M et al (2019) Chronic exposure to environmental concentrations of phenanthrene impairs zebrafish reproduction. Ecotoxicol. Environ. Saf. 182, 109376. https://doi.org/10.1016/j.ecoenv.2019.109376
Pradhan A, Olsson PE (2015) Zebrafish sexual behavior: role of sex steroid hormones and prostaglandins. Behav. Brain Funct. 11, 23. http://10.1186/s12993-015-0068-6
Rose J, Holbech H, Lindholst C et al (2002) Vitellogenin induction by 17β-estradiol and 17α-ethinylestradiol in male zebrafish (Danio rerio). Comp. Biochem. Physiol. C Toxicol. Pharmacol. 131, 531-539. https://doi.org/10.1016/S1532-0456(02)00035-2
Sainz-Cort A, Jimenez-Garrido D, Muñoz-Marron E et al (2021) Opposite roles for cannabidiol and δ-9-Tetrahydrocannabinol in psychotomimetic effects of cannabis extracts: A naturalistic controlled study. J. Clin. Psychopharmacol. 41, 561-570. http://10.1097/jcp.0000000000001457
Singh SK, Srivastava AKJJoE, Health O (2015) Variations in Hepatosomatic Index (HSI) and Gonadosomatic Index (GSI) in Fish Heteropneustes fossilis Exposed to Higher Sub-Lethal Concentration to Arsenic and Copper. 15, 89-93.
Smolinsky AN, Doughman JM, Kratzke L-TC et al (2010) Zebrafish (Danio rerio) androgen receptor: Sequence homology and up-regulation by the fungicide vinclozolin. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 151, 161-166. https://doi.org/10.1016/j.cbpc.2009.10.001
Sullivan CV, Yilmaz O (2018) Vitellogenesis and yolk proteins, fish. In Skinner, M. K. (Eds), Encyclopedia of Reproduction (Second Edition). Oxford, Academic Press: 266-277.
USFDA, 2018. FDA approves first drug comprised of an active ingredient derived from marijuana to treat rare, severeforms of epilepsy. US Food and Drug Administration.
Valim Brigante TA, Abe FR, Zuardi AW et al (2018) Cannabidiol did not induce teratogenicity or neurotoxicity in exposed zebrafish embryos. Chem. Biol. Interact. 291, 81-86. http://10.1016/j.cbi.2018.06.008
Wu X-J, Williams MJ, Kew KA et al (2021) Reduced vitellogenesis and female fertility in gper knockout zebrafish. Front. Endocrinol. (Lausanne) 12. http://10.3389/fendo.2021.637691
Zhang X, Danaceau JP, Chambers EE, 2015a. Quantitative analysis of THC and its metabolites in whole blood using LC-MS/MS for toxicology and forensic laboratories. Waters Application Note 720005556.
Zhang Z, Zhu B, Ge W (2015b) Genetic analysis of zebrafish gonadotropin (FSH and LH) functions by TALEN-mediated gene disruption. Mol. Endocrinol. 29, 76-98. http://10.1210/me.2014-1256

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Cannabidiol Inhibited Reproductive Ability of Zebrafish (Danio Rerio) with Gender-Dependent Manner

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Abstract

Figures

1. Introduction

2. Experimental

2.1 Chemicals

2.2 Zebrafish maintenance

2.3 Acute toxicity test

2.4 Short-term reproduction test

2.5 Zebrafish dissection

2.6 Histological observation of ovary and testicle

2.7 RNA extraction, cDNA synthesis, and qRT-PCR

2.8 CBD detection in zebrafish organs and exposure solution

2.9 Data analysis

3. Results And Discussion

3.1 Chemical analysis

3.1.1 Method validation

3.1.2 CBD analysis in exposure solution

3.2 Acute toxicity of CBD on adult zebrafish

3.3 CBD decreased the reproductive ability of adult zebrafish

3.4 Accumulation of CBD in zebrafish organs

3.5 Effect of CBD on gonad and liver of zebrafish

3.6 Effect of CBD on contents of hormone and Vtg

3.8 Effect of CBD on reproduction-related gene expression

3.9 Effect of CBD on apoptosis-related gene expression

4. Conclusion

Abbreviations

Declarations

References

Supplementary Files

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