Abstract
Although much attention has focused on environmental contamination by heavy metals, pesticides, and polychlorinated biphenyls, potential deleterious effects of naturally occurring organic compounds have received much less consideration. Saponins, which are glycosides found in many plants, are important, environmentally ubiquitous organic compounds. Saponins have both beneficial and deleterious effects in adults, but little is known about how saponins effect early vertebrate embryonic development. The authors tested the toxicity of quillaja saponin using a zebrafish embryo assay. Quillaja saponin, extracted from bark of the tree, Quillaja saponaria, is a common foaming agent used in foods and beverages. At 6 h post fertilization, zebrafish embryos were exposed to five concentrations (0 [negative control], 1, 5, 10 or 20 μg) of quillaja saponin per milliliter of medium. Zebrafish embryos exposed to 2% ethanol were positive controls (100% embryonic death). Embryos were assessed at 30, 54, and 72 h post fertilization for changes in embryonic development, mortality, time of hatching, and morphological deformities. Embryos exposed to 1 and 5 μg saponin were healthy, showed no obvious deformities, but exhibited shrinkage of the chorion. Hatching time for zebrafish embryos exposed to 1 and 5 μg/ml saponin decreased by 18 h compared to unexposed embryos. Zebrafish embryos treated with 5 μg/ml saponin responded less to touch than embryos treated with 1 μg/ml saponin or controls. Zebrafish embryos exposed to more than 5 μg/ml saponin exhibited 100% embryonic mortality. These results indicate that exposure to 5 μg/ml or less of quillaja saponin acts as a growth promoter, whereas concentrations of 10 μg/ml or greater are lethal.
Much of the current research in toxicology has mainly focused on heavy metals, including mercury, cadmium, arsenic, zinc, and copper, and on pesticides and polychlorinated biphenyls (PCBs); less work has been done to elucidate the detrimental effects of various organic compounds (Kohler, Belkins, and Schmid 2000). One extremely important category of organic compound with both beneficial and detrimental consequences of exposure is the saponins, which are found in many plants including most vegetables, beans, (including soybeans) and various herbs. Saponins are glycoside compounds consisting of a hydrophobic core molecule that is attached to one or more sugar side chains at different carbon sites of the hydrophobic core (Higuchi et al. 1987; Higuchi, Tokimitsu, and Komori 1988). Sometimes saponins can taste bitter, reducing palatability of foods. Alfalfa is an important livestock feed that is high in protein but its use as a livestock feed source is limited by the antinutritional influence of saponins also found in alfalfa (Sen, Makkar, and Becker 1998a; Sen et al. 1998b). Many saponins, including those found in alfalfa, reduce the feed intake and growth rate of nonruminant animals, cause hemolysis of red blood cells and some saponins can even result in life-threatening toxicities for certain animal species (Cheeke 1995; Sen et al. 1998a). Quillaja saponin, which contains 8% to 10% saponin based on dry matter weight, is a common commercially available saponin with many deleterious properties including fish poisoning (Mahato, Sudip, and Poddar 1988; Hostettmann and Marston 1995). It contains two sugar side chains located at C-3 and C-28 of the hydrophobic core (Lacaille-Dubois et al. 1999).
Zebrafish (Danio rerio) are a powerful vertebrate animal model system that can be used as an effective alternative to mammalian animal testing to study mechanisms of toxicity (Carvalho, Araújo, and Santos 2006). The many advantages to using zebrafish embryos in toxicity studies include transparent embryos, rapid ex utero development, and simple, accurate chemical delivery (Hill et al. 2003; Gonzalez et al. 2005). In addition, a single pair of zebrafish can reliably produce up to 200 embryos per week. Zebrafish reach sexual maturity in only 3 months, and their genetics and developmental biology are well documented. Zebrafish central nervous system (CNS) development is easy to study. A functioning nervous system is in place by 24 h post fertilization (hpf) and the ability to array embryos into multi-well assay plates allows for testing multiple exposure conditions rapidly and efficiently. Over the past few years, it has become apparent that zebrafish as a model of normal vertebrate development as well as disease pathogenesis has received increased attention by the scientific community, primarily because of its value in both experimental and genetic analyses.
Kimmel et al. (1995) divided zebrafish embryonic development into eight specific stages: zygote, cleavage, gastrula, segmentation, pharyngula, hatching, and early larva. They also determined the exact age of the embryos in each period. Zebrafish embryos develop normally if they are kept in water that is within a range of 25°C to 33°C, with 28.5°C being the optimal temperature for normal development (Westerfield 2000). Zebrafish naturally hatch from their protective egg covering or chorion on day 3 after fertilization (Westerfield 2000).
Often, the beneficial or deleterious side effects of exposure to specific compounds are a function of concentration. The effects of exposure of quillaja saponin on zebrafish survival and development have not been investigated previously. Therefore, we assessed the toxicity of quillaja saponin (Quillaja saponaria) using a zebrafish (Danio rerio) embryo assay. The effects of different concentrations of quillaja saponin were examined with respect to lethality and changes in morphology during the first 3 days of zebrafish development.
MATERIALS AND METHODS
Zebrafish Embryos
Adult fish were raised and kept under standard laboratory conditions at 28.5°C (Westerfield 2000) in the Department of Biology at Texas A&M University. Male and female adult zebrafish were paired in the evening and fertilized embryos were obtained at 10 to 11 <sc>am</sc> the following morning. Medium, consisting of ultrapure water containing low concentrations of specific ions and adjusted to pH 7.2, was used to maintain the developing zebrafish embryos and was freshly prepared for each experiment according to Westerfield (2000). All zebrafish embryos were staged and fixed at specific hours post fertilization (hpf) as described by Kimmel et al. (1995). Both adult and embryonic zebrafish embryos were maintained according to protocols that were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 85-23, revised 1996).
Quillaja Saponin Preparation
Quillaja bark saponin (Sigma-Aldrich, St. Louis, MO, USA) containing 8% to 10% saponin was added to freshly made embryo medium to yield four different concentrations: 1, 5, 10 and 20 μg quillaja saponin/ml. All solutions were made immediately before exposure to zebrafish embryos.
Lethal Dose (LD) Assay
The dose response assay to determine the lethal dose for quillaja saponin was conducted in 24-well flat bottom plates with low evaporation lids (BD Biosciences, San Jose, CA, USA). The total volume of zebrafish medium in each well was 2 ml. Four wells on each plate were used for negative controls and contained only zebrafish medium. Four wells on each plate were used as positive controls and contained zebrafish medium with 2% ethanol (Mindel 2000). The remaining 16 wells were divided into four wells, each containing 2 ml of zebrafish embryo medium and one of four different doses of quillaja saponin, described in the previous section. Two zebrafish embryos were added to each well, the 24-well plates were prepared in duplicate and a total of 96 zebrafish embryos were tested. The 24-well plates were covered with low evaporation lids and incubated for 72 h at 28.5°C (Thelco Laboratory Incubator; Cole-Palmer Instrument, Vernon Hills, IL, USA).
Morphological Assessment
The effects of exposure to different concentrations of saponin on zebrafish embryo morphology were assessed at 30, 54, and 72 hpf, using a SZ-40 binocular microscope (Olympus, Center Valley, PA, USA). Images of embryos at different hpf were captured using an Eclipse E400 microscope equipped with a 2× objective, a DXM1200 digital camera, and ACTI imaging software (Nikon Instruments, Melville, NY, USA). Zebrafish embryos were examined at room temperature (25°C) to monitor the developmental stage, mortality, hatching, response to touch, and the presence of any deformities. Dead embryos were removed at each monitoring time point and all surviving embryos were photographed.
Statistical Analysis
Each treatment group consisted of eight individual values. One-way analysis of variance (ANOVA) was performed to assess differences among concentrations of quillaja saponin expressed as mean ± standard error of the mean (SEM). Significance was set at p ≤ .05 and treatment mean comparisons were made using multiple comparisons Duncan’s difference procedure (Duncan 1955).
RESULTS
Viability and rate of hatching of zebrafish embryos were first assessed at 24 hpf as shown in Table 1. At 24 hpf, none of the embryos exposed to 2% ethanol were alive, but 100% of the control embryos (not exposed to quillaja saponin) and 100% of the embryos exposed to the lowest dose of quillaja saponin (1 μg/ml) were alive. Of the embryos exposed to the next highest dose of quillaja saponin (5 μg/ml), 93.75% were alive, but all embryos exposed to the two highest doses of quillaja saponin (10 and 20 μg/ml) were dead at 24 hpf. There was no change in the percent of live zebrafish embryos at 48 hpf. These data demonstrate that the lethal dose (LD) of quillaja saponin is higher than 5 μg/ml for zebrafish embryos. Surprisingly, 2 out of 16 (12.5%) zebrafish embryos exposed to 5 μg/ml quillaja saponin had hatched from their chorion by 24 hpf and by 48 hpf 93.75% of the embryos exposed to 5 μg/ml quillaja saponin had hatched, as shown in Table 1. At 48 hpf, 81.25% of the embryos exposed to 1 μg/ml quillaja saponin had hatched, but none of the control zebrafish embryos had hatched.
We first performed morphological assessments at 30 hpf, which was approximately one third the way through the pharyngula stage (24 to 48 hpf), named for the period of time when the pharyngeal arches develop (Kimmel et al. 1995). Zebrafish embryos not exposed to saponin or ethanol (i.e., negative-control embryos) exhibited normal age-specific developmental features at 30 hpf, including rapid lengthening of the body; straightening of the body axis from its curvature around the yolk sac, blood vascular circulation is established with a beating heart, initiation of epidermal pigmentation, and the brain consisted of five vesicles but the pectoral fins were not yet visible. No deformities were observed in the head, eyes, yolk sac, trunk, or tail of control zebrafish embryos at 30 hpf, as shown in Figure 1A . Embryos exposed to the two lowest concentrations of quillaja saponin (1 and 5 μg/ml) also displayed normal age-specific morphology, as shown in Figure 1B and C . However, significant shrinkage of the chorion was observed at both concentrations. Exposure of zebrafish embryos to 5 μg/ml quillaja saponin appeared to accelerate embryonic development without the occurrence of morphological deformities. Therefore, the dose of 5 μg/ml quillaja saponin could be considered as a growth promoter for zebrafish embryos, as shown in Figure 1C .
Zebrafish embryos were next observed at 54 hpf, which is near the beginning of the normal hatching stage (48 to 72 hpf) and a time of rapid morphogenetic change. Negative-control zebrafish embryos not exposed to quillaja saponin or ethanol revealed significant development in all primary organ systems, but especially in the head and pectoral fins, as shown in Figure 2A . Embryos exposed to the two lowest concentrations of quillaja saponin (1 and 5 μg/ml) had all hatched from the chorion, whereas none of the embryos not exposed to quillaja saponin had hatched from their chorions by 54 hpf.
Starting at 24 hpf, zebrafish embryos begin to show motor responses to touch as well as spontaneous movement of the body; the tail coiled rapidly followed by a slower relaxation phase (Saint-Amant and Drapeau 1998 Saint-Amant and Drapeau 2000; Drapeau et al. 2002). Negative-control zebrafish embryos and embryos exposed to 1 μg/ml quillaja saponin exhibited normal spontaneous movements and rapidly responded to touch. However, zebrafish embryos exposed to 5 μg/ml quillaja saponin revealed sluggish spontaneous movements and tended to float near the water surface, once they hatched from their chorion. In general, embryos exposed to 5 μg/ml quillaja saponin were much less responsive to touch than 1 μg/ml quillaja saponin–treated or negative-control embryos.
DISCUSSION
Increased public concern over environment pollution from industrial and agriculture sources has encouraged scientists to explore cost and time-effective alternatives to current chemistry-based methods in detecting pollutants. To this end, developing zebrafish embryos are becoming an important high-throughput vertebrate animal model to test for potential toxicants and to study mechanisms of toxicity. In this study, we observed that exposure of developing zebrafish embryos to low concentrations of quillaja saponin accelerated development, whereas higher concentrations were lethal. A very narrow window for dose of exposure exists between enhanced growth (1 and 5 μg/ml) and significant lethality (10 μg/ml or higher) for quillaja saponin.
Saponins are a large class of compounds that are widely distributed in both wild plants and cultivated crops (Shimoyamada et al. 1990; Price, Johnson, and Fenwick 1987; Westendarp 2005; Mahato, Sudip, and Poddar 1988), in lower marine animals, and even in some bacteria (Riguera 1997; Yoshiki, Kudou, and Okubo 1998), but are uncommon in animal tissues (Hashimoto 1979). The observed toxic effects of quillaja saponin on zebrafish embryos at concentrations of more than 5 μg/ml might be due to physicochemical (surfactant) properties (Güçlü-Ustündag and Mazza 2007) and/or membranolytic effects (Price, Johnson, and Fenwick 1987) on the chorion, which is a semipermeable protective membrane that surrounds the developing embryo until hatching at 48 to 72 hpf. Many studies have reported that quillaja saponin affects animal cell membrane permeability by forming pores in membranes (Authi et al. 1988; El Izzi et al. 1992; Choi et al. 2001; Menin et al. 2001; Plock, Sokolowska-Kohler, and Presber 2001) or by creating pores of 40 to 50 Å diameter in cell membranes of human erythrocytes (Seeman, Cheng, and Iles 1973). It is possible that the zebrafish chorion or the embryo proper is not significantly affected by exposure to low concentrations (1 or 5 μg/ml) of quillaja saponin, because no alterations in morphology were observed in embryos exposed to these concentrations of quillaja saponin. On the other hand, early hatching could be due to weakening or disruption of chorion membrane integrity by quillaja saponin. Some studies suggest that effective embryonic exposure to certain chemicals requires dechorionation of zebrafish embryos (Hammerschmidt, Blader, and Strähle 1999). However, in the present study, embryos treated with quillaja saponin at concentrations more than 5 μg/ml medium were lethal without dechorionating zebrafish embryos, indicating that the toxic effects of quillaja saponin might focus on the semipermeable protective chorion that surrounds the developing embryo. Alternatively, quillaja saponin might be able to easily permeate the noncellular chorion and directly affect cells in the embryo bodies.
Zebrafish embryos exposed to 1 and 5 μg/ml quillaja saponin showed accelerated embryonic development with respect to hatching time compared to nonexposed negative-control embryos. This study is the first record to show that quillaja saponin has both beneficial and harmful effects on zebrafish embryos depending on the concentration. The growth promotion effect of quillaja saponin might be due to induction of increased absorption of ions from the medium. Many saponins can facilitate absorption of certain molecules from the surrounding water such as calcium and iron salts (Freeland, Calcott, and Geiss 1985; Lower 1984; Kimata, Nakashima, and Kokubun 1983, Yata et al. 1985), leading to a wide variety of possible effects, including increased rates of cell proliferation or differentiation. Saponins also are able to alter sodium-potassium ATPase and calcium-magnesium ATPase activity (Choi et al. 2001) by interacting with membrane cholesterol and removing it from the immediate environment of the ATPases. Reductions in membrane cholesterol would lead to increased membrane fluidity, which would facilitate conformational changes that ATPases might undergo during their transport cycle. Membrane fluidity also controls the enzyme activities associated with biological membranes and has an important role in ion transport (Ma and Xiao 1998). Conversely, Brain, Hadgraft, and Al-Shatalebi (1990) reported that insertion of saponins into the cell membrane lipid bilayer is independent of the presence of cholesterol. Hu, Konoki, and Tachibana (1996) also demonstrated that saponins were able to induce a permeability change in liposomal membranes without the presence of cholesterol. Thus, the mode of action of saponin on cell membranes is still unclear and more research is needed to clarify this point.