Amaranthus hybridus (Amaranthaceae) prevents the detrimental effects of cyclophosphamide on ovarian function in Wistar rats: An experimental study

Abstract Background Cyclophosphamide (CP) is an anticancer agent, but its chronic administration induces ovarian toxicity. Objective We evaluated the effects of aqueous extract (AE) and methanol extract (ME) of Amaranthus hybridus (A. hybridus) on CP-induced ovarian toxicity in rats. Materials and Methods 40 female Wistar rats (10 wk, 170-200 gr) were distributed into 8 groups (n = 5/each) as follows: 1) healthy control; 2) CP+distilled water (10 ml/kg/d); 3) CP+3%-tween 80 (10 mL/kg/d); 4) CP+clomiphene citrate (2 mg/kg/d); 5, 6) CP+AE of A. hybridus (55 and 110 mg/kg/d); and 7, 8) CP+ME of A. hybridus (55 and 110 mg/kg/d). After 28 days of treatment, estrus cyclicity, ovarian and uterine weights as well as estradiol levels and ovarian histology were determined. Results CP induced ovarian toxicity after 28 days of exposure. More specifically, CP disturbed the estrus cycle, decreased ovary and uterus weights (p = 0.04), and the 17-β estradiol level (p = 0.04), and induced severe ovarian damages. Remarkably, A. hybridus significantly increased (p = 0.03) the ovarian weight (AE and ME at all doses) and uterus weight (ME at 110 mg/kg/d), compared with the CP-treated rats. Moreover, the 17-β estradiol level was significantly elevated (p = 0.02) in rats given clomiphene citrate and A. hybridus (AE 110 mg/kg/d; ME 55 mg/kg/d). Finally, the ovaries of rats given plant extracts had many corpus luteum and normal follicles, and no cystic follicles. Conclusion prevented the detrimental effects of CP on ovarian function, which could support its traditional use as a fertility enhancer.


Introduction
Cyclophosphamide (CP) is a cytotoxic agent commonly used to treat several types of cancers in children, adolescents, and adults (1). However, this chemotherapy negatively affects the female reproductive system and fertility, especially in younger patients under long-term CP treatment (2,3). Chronic administration of CP alters the primordial follicles (ovarian reserve) and damages the growing ovarian follicles through a double mechanism. Firstly, by interacting with DNA and stimulating cellular apoptosis and cell death (4), it leads to the destruction of ovarian reserve follicles and inhibition of follicular maturation (5). CP also induces an overproduction of reactive oxygen species (ROS), which react with lipids, proteins, and cellular nucleic acids and inhibit steroidogenesis in ovarian cells, causing infertility (4). This reproductive toxicity of CP is mainly attributed to acrolein, an active metabolite of CP responsible for its side effects (5).
In female rats, reproductive toxicity is characterized by estrus cycle disruption, ovarian atrophy, presence of multiple cystic follicles, low 17-β estradiol (E2) concentration in the follicles as well as ovarian ultrastructure lesions (6). Modern treatment of reproductive toxicity involves the use of various drugs such as antioxidant agents and ovulation inductors, but it is commonly associated with severe side effects (7). Medicinal plants are extensively used in the treatment of reproductive toxicity, due to their biosafety, availability, and rich diversity of phytochemical compounds. Previous works have reported the efficacy of some herbal remedies on the prevention/treatment of CPinduced reproductive toxicity in female rats. For instance, the preventive effects of Spirulina sp. (8) and Nigella sativa (9) on CP-induced ovarian damages have been reported. In addition, curcumin (10) and Bushen Huoxue recipe (11) can alleviate the detrimental effects of CP on ovarian function by improving sex hormone levels, antioxidant enzymes, and ovarian reserve.
Amaranthus hybridus (A. hybridus) is a tropical plant commonly used as a food in the culinary arts and as a drug in folk medicine. In Cameroon, its leaves and seeds are used as fertility booster but no scientific report on this property has been published. Various phyto-components such as tannins, phenols, alkaloids, saponins, steroids, and triterpenes have been identified in this plant (12). Previous experiment studies revealed the antimicrobial (12), antidiabetic (13), anticancer (14) and antioxidant (15)

Estrus cycle monitoring and selection of rats with a regular estrous cycle
Twenty-five days prior to the treatment, vaginal smears were collected daily (9:00-10:00 AM) using a glass pipette. The smear was placed on a slide, fixed with methanol, stained with methylene blue (0.03%), dried, and examined microscopically with a 10× objective (Zeiss, X10) (16). Cell descriptions were used to classify the rats based on the stages of their estrus cycle as reported previously (17). Animals with a normal (regular) estrus cycle for at least 3 consecutive cycles were selected for the experiment.
All drugs were orally administered daily for

Intra-ovarian estradiol concentrations and histology
From each animal, 1 ovary was used for ELISA and the contralateral for histology.
After euthanasia, ovaries were crushed and homogenized in 0.9% NaCl (at 5%). The supernatant obtained after centrifugation (3000 × g for 10 min) of the homogenate was used to quantify E2 levels using an ELISA kit (Accubind, Monobind Inc. Lake Forest, USA) as carried out by Ndeingang et al. (17). Calibration of the curve was constructed after reading the absorbance at 450 nm. The absorbance was read within 5 sec.
The standard for the determination of estradiol was prepared from standard stock solutions containing E2 at concentrations of 0, 10, 30, 300, and 1000 pg/ml (17).
Histological study of ovaries was done following a standard procedure. Briefly, samples were dehydrated in alcohol, embedded in paraffin, and sectioned. Ovary sections (5 μm thick) were stained with haematoxylin/eosin and microscopically examined using a light microscope (OLYMPUS, X200) at a magnification of x200. Mature follicles that contained antrum, oocytes, granulosa cells, and basement membrane were classified as normal (18).

Statistical analysis
Data were expressed as mean ± standard error of the mean (SEM). The differences between groups were analyzed by ANOVA, followed by Tukey's test using the STATISTICA software (version 8.0, StatSoft, Inc., Tulsa, USA). P ≤ 0.05 was deemed statistically significant.  (Table I).

Effects of the aqueous and methanol extracts of A. hybridus on ovarian and uterine weights
The ovarian and uterine relative weights were significantly (p = 0.04) lowered after CP treatment, compared to the control (Figure 1). However, the aqueous and methanol extracts of A. hybridus significantly increased (p = 0.03) the ovarian relative weight compared to the CP+DW and CP+TW groups ( Figure 1A). The uterine relative weight was also significantly (p = 0.04) elevated in rats given the methanol extract of A. hybridus at 110 mg/kg, compared with the CP+TW group ( Figure 1B). The aqueous extract of A. hybridus (55 mg/kg) was more effective in modulating the ovarian and uterine relative weights ( Figures 1A   and 1B).

Effects of the aqueous and methanol extracts of A. hybridus on ovarian histology
In the control group, normal ovary architecture was visible. This was illustrated by the presence

Discussion
The main objective of this study was to investigate the preventive properties of aqueous and methanol extracts of A. hybridus on CP-induced ovarian toxicity in rats. CP is commonly used as an anticancer agent (20), but this chemotherapy is associated with various side effects, such as hemorrhagic cystitis, hair loss, bone-marrow toxicity, and reproductive toxicity (4). In addition,  follicles in CP-exposed female rats have been noted by other research teams (10,30

Conclusion
CP treatment induced ovarian toxicity, characterized by estrus cycle disruption, low ovarian and uterus weights as well as a decline in E2 levels and severe ovarian structural damages. These reproductive, hormonal, and structural alterations were prevented by A.
hybridus extracts. These results could further support the traditional use of A. hybridus as a fertility agent.