Reproductive toxicity assessment of Senecio scandens extract in kunming mice

Genus Senecio (family Asteraceae) has phytochemicals that can influence folliculogenesis and steroidogenesis, which affect reproduction (Mbemya et al., 2017). Senecio scandens Buch.-Ham. is a common species used in folklore medicines in Asia (Wang et al., 2013).

However, phytotoxins such as pyrrolizidine alkaloids (PAs) have been identified in S. scandens (Li et al., 2008b). Toxins affect the reproductive health of humans and animals (Green and Christie, 1961; Khera et al., 2016). To allay any fears and promote the use of S. scandens, analytical procedures for extractions of safe compounds and analyses of PA composition in SSEs are reported (Xiong et al., 2014). Previous studies from our laboratory found SSE to be less toxic in mice based on bone macronucleus rate and acute toxicity study (Chen et al., 2007; Li et al., 2008a). SSE is suggested to be less toxic based on hepatotoxic evaluations (Li et al., 2008a; Xiong et al., 2014).

This study aimed to evaluate the reproductive toxicity of SSE in mice using a three-phase reproductive toxicity evaluation and sperm abnormality rate. The procedure is consistent with the guidelines for preclinical safety evaluation of a new drug and the International Council for Harmonization of Technical Requirements for Pharmaceuticals for human Use (ICU) guidelines (Han et al., 2014a; Xue et al., 2015). Potential adverse effects of S. scandens on reproductive function, embryonic development and postnatal development were assessed (Han et al., 2014a; Xue et al., 2015). The study would also provide guidelines for safe clinical use of S. scandens at reproductive stages in humans and animals.

Animal experiments were conducted according to Animal Research Reporting of in vivo Experiment (ARRIVE) guidelines. The experimental protocol was approved by Guangdong Ocean University Animal Ethics Committee (approval no. 20160137).

Plant shoots were collected from Lipu County, Guangxi Province, China and identified as Senecio scandens Buch.-Ham. by the Institute of Agricultural Biology at Guangdong Ocean University, Zhanjiang, China. The shoots were dried and made into a powder. Each 20-g lot of S. scandens powder was extracted for 8 h with 600 mL of aqueous ethanol (60%), using Soxhlet apparatus. The ethanolic extract was filtered and evaporated to dryness using a rotary evaporator (Model RE52; Shanghai Huxi Instruments) with a benchtop freeze dryer (Virtis Corporation, USA). LD50 of the extract was first determined with a modified Karber’s method (Karber, 1931) and it was estimated to be 3,911±153 mg/kg (95% confidence interval).

A total of 300 (150 male and 150 female), healthy 8-10 week old (sexually matured) Kunming mice (body weight 18-22 g) were purchased from the Guangdong Medical College experimental animal center (certificate number: GDZJ2016A028) and acclimatized for 1 week prior to the start of the experiment.

Four experiments were conducted; general reproductive toxicity (Experiment 1), teratogenicity (Experiment 2) and perinatal development toxicity (Experiment 3) and sperm abnormality rate (Experiment 4) (Table 1).



All test mice were cage-mated overnight in 1:1 mating ratio and the gestation age was calculated from the day of the vaginal suppository test. Mice were provided commercial rodent chow and sterile water ad libitum and housed in polypropylene cages at a temperature range of 23-26°C, 50-55% humidity and a 12-h light/dark cycle. Mice were observed cage-side throughout the experiment.
 
General reproductive toxicity study (Experiment 1)
 
Male mice were injected for 60 days and female mice for 14 days prior mating. Female mice continued to receive treatment dosages from gestation date (GD) 0 to GD 6, during the pre-embryonic implantation stage. Female mice were sacrificed on GD 19 post-organogenesis. The uterus was removed from the abdominal cavity and the numbers of live and dead fetuses recorded. Body weight, body length, tail length and sex of each fetus were also determined and recorded.

External abnormalities such as frontal fracture, small eye, bulky eye or no eye, single nostril or nostril enlargement, no ear, no jaw, short limbs, deformed feet were recorded. Fetuses were then subjected to a visceral and skeletal malformation examination using differential staining as described as follow.

Examination for skeletal malformations was conducted according to Zhao et al., (2010) with minor modifications. Fetuses were eviscerated, skinned and placed in ethanol for 3 days and then double-stained with Alizarin red S and Alcian blue solution for 3 days. After staining, the fetuses were rinsed with deionized water and placed in macerating solution (0.75% KOH) for 3 days. Next, the fetuses were rinsed again with deionized water and placed in a clearing solution (70% ethanol, 100% glycerin and 100% benzyl alcohol) for 24 h. The fetuses were kept in 80% glycerol, and skull, occipital bone, ribs, sternum, vertebrae, limb bones and pelvis were examined under magnifying lens. In each group, the number of fetuses with deformities was expressed as a percentage of the total number of fetuses examined per group.

The fresh fetus dissection technique described by Stuckhardt and Poppe (1984) was followed with slight modifications. Organs in the abdomen, pelvic and thoracic regions were identified and abnormalities examined using a magnifying lens. Numbers of fetuses with deformities in each group were counted and expressed as a percentage of the total number of fetuses examined in that group.
 
Teratogenicity study (Experiment 2)
 
Female mice were administered SSE from GD 6 to GD 15, the organogenesis period. Maternal body weight was recorded every 3 days from GD 0 to GD 18. After parturition, the number of live litter, number of dead litter, litter body weight, litter body length and litter tail length were determined and recorded. External deformity, skeletal deformities and visceral deformities were examined as in Experiment 1.
 
Perinatal toxicity study (Experiment 3)
 
Female mice were administered SSE from GD 15 to 21 days post-parturition. Litter size, external deformity rate and number of litter surviving at 21 days post-parturition were determined. In each treatment group and control group, 20 Fl mice (70-day-old) were coupled. Mice from each group were cage-mated overnight in a sex ratio of 1:1. Pregnancy rate, gestation age, litter size, litter weight and congenital abnormality rate were observed. Anal-genital distance, age at which eyes of pups opened and post-natal death of pups were observed.
 
Sperm abnormality study (Experiment 4)
 
Male mice were injected with SSE at doses of 130, 391 and 1,303 mg/kg/d respectively for 30 days. Mice in the negative control group were injected with 0.6 mL of sterile water and mice in the positive control group were injected 40 mg/kg/d of 0.1% cyclophosphamide. Mice were sacrificed by cervical vertebrae dislocation, 6 h after the last dose. Both right and left epididymis were excised and washed with normal sterile and minced with a microsurgical scissor to form a homogenous suspension. Cells were stained with a mixture of normal sterile and 1% eosin (9:1) for 45 min. Smears were prepared on clean, grease-free slides. The slides were air-dried and coded for subsequent microscopic examination. Cytological evaluation for morphological abnormalities was carried out using an optical microscope at 1000´ magnification. For each male mouse, 200 sperms were assessed. The sperms were classified as normal or abnormal according to WHO guidelines for sperm morphology assessment (Natali and Turek, 2011).
 
Statistical analysis
 
Data were analyzed using ANOVA (SAS Institute 2001 software) and expressed as mean±standard deviation. Kolmogorov-Smirnov goodness of fit test (K-S test) was used to check the assumption of normality. Duncan’s multiple range test was used to compare differences between means of groups. Probability values were considered significant at P<0.05.

Table 2 shows the effects of SSE on the general reproductive functions of mice. Pre- and early- pregnancy administration of SSE at <391 mg/kg/d had no adverse effect on sexual behavior and reproductive functions and no marked anti-androgenic effect on sex ratio. In contrast, flavonoid and phenolic compounds caused adverse effects on olfactory sensitivity and “perceptual block” of stimuli, and also had anti-androgenic effects in mice and rats (Adeiza and Minka, 2011; Rashed et al., 2014). The result of this experiment therefore suggests that <391 mg/kg/d of SSE may have lower amounts of such polyphenols to induce general reproductive toxicity in mice.

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The toxic effects of SSE on mice during the teratogenic sensitive period (Experiment 2) are shown in Table 3 and 4 and Fig 1. Less than 391 mg/kg/d of SSE did not cause maternal toxicity or fetal toxicity but rather enhanced body weight during the organogenesis period and mice gained weight at the end of the dam organogenesis period. In contrast, >30 g/kg body weight of water extract, total alkaloids and a Chinese herbal formula (Qianbai Biyan Pian) from S. scandens caused embryo toxicities in Sprague-Dawley rats when orally administered in the teratogenic sensitive period (Zhao et al., 2010). Seneciphylline and sirkinine also caused embryotoxicity in mice in a whole embryo culture at >12.5 µg/mL (Han et al., 2014b). However, their concentration in S. scandens has been reported to be insignificant to induce toxicities (Xiong et al., 2014). Li et al., (2008a) further observed that, these PAs are eliminated through ethanol extraction in SSE (Li et al., 2008a). SSE therefore could not induce toxicity at the teratogenic sensitive period. It could regulate redox homeostasis to overcome oxidative stress at pregnancy to enhance maternal and fetal development (Staicu et al., 2011).

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The effects of SSE on perinatal development toxicity are shown in Table 5 and 6. The pregnant mice showed no abnormality and the weight gain of each group was similar. There was no significant difference in litter size between treatment groups and control. Litter sizes ranged from 7 to 14 pups per dam. Anal-genital distance, age at which eyes of pups opened and post-natal death of pups were within the normal ranges. Neonates are highly susceptible to phytotoxins due to the potential synergy with their high liver copper levels (Wang et al., 2013). Postnatal exposure to phytoestrogens can also induce endocrine disruptions and causes adverse reproductive and neurobehavioral effects (Ryokkynen et al., 2006). Terpenoids, flavonoids, and their glycosides from S. scandens are estrogenic (Wang et al., 2013; Shrestha et al., 2014). Higher doses of phytoestrogens induced adverse effect on brain aromatase and neurobehavioral activities but lower doses did not (Setchell et al., 1997; Wisniewski et al., 2005). In this study, <391 mg/kg/d of SSE caused no marked perinatal development toxicity in mice. It thus suggests <391 mg/kg/d of SSE may have lower phytoestrogens concentrations to induce perinatal development toxicity in mice.

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Sperm abnormality rate is a reliable method of evaluating genetic reproductive toxicity in male rodents (Ramachandran and Singh, 2017). Effect of SSE on sperm abnormality rate is shown in Fig 2. From the study, <391 mg/kg/d of the extract induced no increase in sperm abnormality. This is in agreement with Chen et al., (2007) findings that <390.8 mg/kg/d of S. scandens ethanol extract had no adverse effect based on the bone marrow micronucleus test. However, SSE showed a dose- response effect and the potential to induce genetic reproductive toxicity at <1,303 mg/kg/d. Sperm shape might have been impaired at <1,303 mg/kg/d of SSE due to peroxidation of polyunsaturated fatty acids (PUFAs) in spermatozoan membranes (Sanocka and Kurpisz, 2004; Natali and Turek, 2011; Xie et al., 2014). Phenolic compounds and flavoniods present in SSE are reported to act as pro-oxidants at high doses (Eghbaliferiz and Iranshahi, 2016). Liu and Ng (2000) observed that S. scandens water extract caused pro-oxidation and DNA damage due to higher amount of flavonoids and phenolic compounds. It is interesting knowing that, the genetic reproductive toxicity of SSE at 1,303 mg/kg/d was due to increasing doses of phenolic compounds and not PAs.