Spirulina ( Arthrospira ) Protects Against Cadmium-Induced Teratogenic Damage in Mice

Abstract

The role of Spirulina (Arthrospira) in preventing cadmium (Cd) teratogenicity in ICR mice was studied. Cd was administered intraperitoneally to female mice at 1.5 mg/kg on gestation day (GD)-7, and Spirulina was given by peroral (intragastric) administration at 62.5, 125, 250, or 500 mg/kg from GD-0 through GD-17 (the day when animals were sacrificed). Because among the mechanisms suggested to account for reproductive damage are oxidative stress and lipoperoxidation, embryonic hydroperoxides were also determined. Treatment with Spirulina at the three highest doses significantly decreased the frequency of fetuses with exencephaly, micrognathia, and skeletal abnormalities induced by Cd. Furthermore, Spirulina treatment significantly and dose-dependently decreased lipid peroxidation, which was dramatically increased by administration of the metal. The results of the present study clearly point to the therapeutic potential of Spirulina in Cd-induced teratogenicity and probably through its antioxidant activity.

Introduction

C admium (Cd) is an important industrial and environmental pollutant widespread in the biosphere^1,2 that can produce a wide variety of adverse effects in humans and animals.³ Cd is perhaps the most widely studied elemental teratogen, although its mechanism of action is poorly understood.⁴ It has been found to induce teratogenicity in rats,^5
–7 hamsters,⁸ mice,⁹ frogs,¹⁰ and chickens.¹¹ If mouse embryos are exposed to Cd before neurulation, the most dramatic malformation observed is an opening in the anterior neural pore,^12,13 although other abnormalities such as microphthalmia, heart defects, malformations of the face,¹⁴ and delayed ossification¹⁵ have also been observed.

It has also been demonstrated that Cd increases the embryolethality caused by herbicides and insecticides in chicken embryos¹⁶ and produces damage in the male reproductive system of some species of rodents.^17
–19

Results of in vivo and in vitro studies demonstrate that Cd induces an oxidative deterioration of biological macromolecules^20,21 and that embryotoxicity is partly due to oxidative DNA changes associated with increased production of reactive oxygen species, decreased antioxidant enzyme levels, and interaction with the enzymes that repair damaged DNA.^22,23

Spirulina, which belongs to the cyanobacteria (blue-green algae group), is potentially of considerable importance in human and animal nutrition.²⁴ It is a rich source of proteins, vitamins, essential amino acids, minerals, essential fatty acids, glycolipids, sulfolipids,²⁵ phycobilins, and other phytochemicals.²⁶

Some pharmacological activities of Spirulina have been previously reviewed in other publications.^24,27

–30 Spirulina has also been shown to have antigenotoxic activity^31

–35 and antitoxic properties against effects of metals, pharmaceuticals, and radiations.^36

–40 Some of these effects may be attributed to the antioxidant activity of Spirulina itself,^41,42 of its extracts,^43,44 or of some of its components such as phycocyanin,^45
–47 the latter of which constitutes about 14% of the total dry weight of Spirulina.⁴⁸ SP is also reputed to be an external source of the vital antioxidant enzyme superoxide dismutase.^37,49

On the basis of these reports, in the present study we focused on determining whether Spirulina would protect against Cd-induced teratogenicity when given before neurulation in ICR mice and, if it does, the relation with the antioxidant effect.

Materials and Methods

Materials

Cd (as CdCl₂) was of analytical grade and was purchased from Sigma Chemical Co. (St. Louis, MO, USA). The Spirulina sample was from a bulk production batch (5DW-9714) of standardized quality, supplied by Alimentos Esenciales para la Humanidad, S.A. de C.V., Mexico City, DF, Mexico.

Experimental setup

Experimental animals were dealt with according to institutional guidelines and the Mexican Official Standard (NOM-062-ZOO-1999) regarding technical specifications for reproduction, care, and use of laboratory animals.

ICR female and male mice (Harlan of Mexico, DF) weighing 30 ± 2 g were used. Mice were kept in a room at a constant temperature of 22 ± 1°C and 12-hour light/dark cycles (light on from 9:00 to 21:00 hours) and given Purina (St. Louis) Rodent Lab Chow and water ad libitum. Males were housed individually, but females were kept two per cage. All cages were provided with a sawdust bed. Timed matings were produced by placing individual male mice into cages containing two nulliparous females for 3 hours of the dark cycle, 6:00 to 9:00 a.m. The presence of a vaginal plug was regarded as evidence of coitus, and this day was designated as gestational day (GD)-0.

Pregnant females were randomly divided in seven groups of animals, as follows: Group I, control (saline); Group II, 1.5 mg of CdCl₂/kg of body weight; Group III, 500 mg of Spirulina/kg of body weight; Group IV, 62.5 mg of Spirulina plus 1.5 mg of CdCl₂/kg of body weight; Group V, 125 mg of Spirulina plus 1.5 mg of CdCl₂/kg of body weight; Group VI, 250 mg of Spirulina plus 1.5 mg of CdCl₂/kg of body weight; and Group VII, 500 mg of Spirulina plus 1.5 mg of CdCl₂/kg of body weight.

CdCl₂ was dissolved in saline solution and administered to pregnant mice by intraperitoneal injection on GD-7. The 1.5 mg/kg dose was found to produce exencephaly in 60–80% of fetuses in a previous study.⁵⁰

Spirulina suspended in saline was given to mice daily by peroral (intragastric) administration during the whole gestational period (GD-0–GD-17). In this case the doses were selected on the basis of previous studies, where it was demonstrated that the largest dose was very well tolerated by the mice when administered by this route.³⁵

Evaluating dams and litters

Pregnant mice were sacrificed by cervical dislocation on GD-17. The uterus of each animal was removed, and the number of implantation sites, resorptions, and viable fetuses were recorded. Body weight and gross malformations of fetuses were recorded. One-third of the offspring were fixed in 95% alcohol for skeletal staining according to Peters' double-staining method for skeletal malformations and variations.⁵¹ The remaining fetuses were fixed in Bouin's solution for 2 weeks and prepared for serial slices.⁵² They were examined under a Zeiss (Carl Zeiss of Mexico, DF) dissection stereomicroscope.

Evaluating lipoperoxidation

Pregnant mice were randomized into four groups of five dams each: Group I, control; Group II, Spirulina (500 mg/kg dose); Group III, CdCl₂ at 1.5 mg/kg; and Group IV, CdCl₂ at 1.5 mg/kg + Spirulina (500 mg/kg). Spirulina was administered daily from GD-0 to GD-10 by peroral (intragastric) administration; CdCl₂ was injected intraperitoneally on GD-7. All animals were sacrificed by cervical dislocation on GD-10, and a hysterectomy was performed to obtain the embryos. Three embryos from each litter were tested for lipid peroxidation by measuring the micromolar hydroperoxide concentration with a Calibiochem kit (Sigma).

Statistical analysis

The litter was used as the experimental unit for maternal parameters (implantations, resorptions, live fetuses, placental diameter, and placental weight). The fetus was the experimental unit for the malformation parameters. The χ² test followed by Fisher's exact test was used to evaluate fetuses with abnormalities as well as the type of external and skeletal anomalies. The remaining variables were assessed by one-way analysis of variance and then by Student–Newman–Keuls test (post hoc test) for paired comparisons.

Results

Teratological examination

Fetuses from females treated with CdCl₂ at 1.5 mg/kg on GD-7 exhibited malformations (Table 1). Placental weight was increased, but none of the other variables, such as maternal weight (data not shown), implantations, and resorptions per mother, was altered in comparison with the control group. Administration of Spirulina alone induced no teratogenic effect. The protection with Spirulina had a dose-dependent effect on the number of fetuses with external malformations, mainly in relation to exencephaly and micrognathia.

Table 1.

Effect of Cadmium and Spirulina Treatment on Gestational Parameters in ICR Mice

				Fetal		Placental
Treatment (mg/kg)	Number of pregnant females	Implantations sites/dam	Resorptions/dam	Crown–rump length (cm)	Weight (g)	Diameter (cm)	Weight (mg)
Control saline	10	14.1 ± 0.7	0.2 ± 0.1	2.00 ± 0.01	0.9 ± 0.01	0.72 ± 0.005	10 ± 0.06
CdCl₂ (1.5)	10	14.9 ± 0.7	3.3 ± 1.0^a	1.90 ± 0.01	0.80 ± 0.01	0.75 ± 0.006	11 ± 0.09
Spirulina
500 alone	10	14.8 ± 0.5	1.0 ± 0.3	1.98 ± 0.01	0.94 ± 0.01	0.71 ± 0.005	9 ± 0.01
62.5 + CdCl₂	10	14.7 ± 1.1	3.1 ± 0.9	1.92 ± 0.01	0.78 ± 0.01^a	0.67 ± 0.006^b	8 ± 0.01^b
125 + CdCl₂	10	15.1 ± 0.6	2.3 ± 1.0	1.94 ± 0.01	0.90 ± 0.01	0.71 ± 0.005^b	10 ± 0.06
250 + CdCl₂	10	14.7 ± 0.8	0.4 ± 0.2^b	1.97 ± 0.01^b	0.90 ± 0.01	0.70 ± 0.003^b	9 ± 0.01
500 + CdCl₂	10	15.5 ± 0.6	1.0 ± 0.4	1.97 ± 0.01^b	0.97 ± 0.01	0.70 ± 0.004^b	10 ± 0.05

Data are mean ± SE values.

Significantly different (P < .05) from ^acontrol, ^bCdCl₂ (1.5).

CdCl₂, cadmium chloride.

The incidence and type of each of the external malformations are shown in Table 2. Mothers treated with Spirulina at 62.5, 125, 250, and 500 mg/kg on GD-0 to GD-17 and CdCl₂ at 1.5 mg/kg on GD-7 had fewer fetuses with abnormalities compared to the CdCl₂ group. The malformations produced by Cd included principally exencephaly, micrognathia, and open eye (Table 2). No additional malformations were detected by visceral examination.

Table 2.

Incidence and Type of External Malformations in ICR Mice Treated with Spirulina and Cadmium

			Number of fetuses with abnormalities
Treatment (mg/kg)	Number examined	Number of abnormal fetuses	Exencephaly	Micrognathia	Open eye	Clubfoot	Celosomy
Control (saline)	139	0	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)
CdCl₂ (1.5)	114	73^a	68 (59.6)^a	62 (54.4)^a	18 (15.8)^a	1 (0.9)	0 (0)
Spirulina
500 alone	137	0	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)
62.5 + CdCl₂	118	6	68 (57.6)	68 (57.6)	17 (14.4)	6 (5.18)	3 (2.5)
125 + CdCl₂	128	54^b	43 (33.6)^b	40 (31.2)^b	5 (3.9)^b	6 (4.7)	2 (1.6)
250 + CdCl₂	142	18^b	10 (7.0)^b	3 (2.1)^b	0 (0)^b	2 (1.4)	0 (0)
500 + CdCl₂	145	2^b	2 (1.4)^b	1 (0.8)^b	0 (0)^b	0 (0)	0 (0)

Data are mean ± SE values (%).

Significantly different (P < .05) from ^acontrol, ^bCd (1.5).

Regarding skeletal anomalies, the most striking effect was seen on the offspring of the CdCl₂ group, including reduced ossification of the head and vertebrae, as well as fused ribs (Table 3). Spirulina at the highest doses significantly (P < .05) decreased the frequency of these alterations, with this protection being more evident at 500 mg/kg.

Table 3.

Effects of Maternal Exposure to Cadmium and Spirulina on Fetal Mouse Skeleton Morphology

		Number of fetuses with reduced ossification of
Treatment (mg/kg)	Number	Head	Sternebrae	Vertebrae	Fused ribs
Control (saline)	45	0 (0)	0 (0)	0 (0)	0 (0)
CdCl₂ (1.5)	40	29 (72.5)^a	29 (72.5)^a	20 (50)^a	14 (35)^a
Spirulina
500 alone	40	0 (0)	0 (0)	0 (0)	0 (0)
62.5 + CdCl₂	37	24 (64.9)	32 (86.5)	13 (35.1)	6 (16.2)^b
125 + CdCl₂	39	19 (48.7)^b	22 (56.4)^b	5 (12.8)^b	2 (5.1)^b
250 + CdCl₂	49	7 (14.3) ^b	18 (36.7)^b	0 (0)^b	2 (4.1)^b
500 + CdCl₂	50	1 (2.0)^b	22 (44.0)^b	0 (0)^b	1 (2.0)^b

Data are mean ± SE values (%) values. Head refers to the calvaria.

Significantly different (P < .05) from ^acontrol, ^bCdCl₂ (1.5).

Lipid peroxidation

In GD-10 embryos the level of lipid peroxidation measured by the hydroperoxide concentration was significantly increased by 160% (from 50 to 85 μM) in the CdCl₂-treated group, whereas in the group treated with Spirulina at 500 mg/kg + CdCl₂ it decreased by 82% (from 200 to 150 μM) (P < .05), more than three times the control values (Fig. 1). The health of dams was not affected by Cd or Spirulina administration.

FIG. 1.

Lipid hydroperoxide content in embryos exposed to Spirulina (SP) and CdCl₂ during gestation. By analysis of variance, Holm-Sidak test: ^a P < .05 versus control and SP groups, ^b P < .05 versus CdCl₂ alone group.

Discussion

Intraperitoneal administration of CdCl₂ at a dose of 1.5 mg/kg to ICR mice on GD-7 produced micrognathia, open eye, and principally exencephaly, showing the possibility that certain cells in the embryo are exceptionally sensitive to low levels of a teratogenic agent.⁵³ These results coincide with numerous studies in which Cd exposure before neurulation caused an opening in the anterior neural pore.¹³ However, the frequencies of such alterations were different from those reported by other authors, which can be explained by the dose, route of administration, gestational age, species, and animal strain^3,13 and the importance of genetic background in determining sensitivity. On the other hand, the mechanisms of action are as yet unknown.⁵⁴

Cd induced cellular glutathione and thiol depletion, which may cause enhanced production of reactive oxygen species, resulting in increased lipid peroxidation, modulation of intracellular oxidized states, DNA damage, membrane damage, altered gene expression, and apoptosis.^20,55,56

No significant effect on fetal or placental weight was observed in the control and experimental groups treated with Spirulina. With CdCl₂ there was a tendency to an increase in placental diameter, which suggests that the embryotoxicity observed here may include a placental lesion.⁵⁷ The combined treatment of Spirulina and Cd significantly decreased this alteration, although more studies are needed to confirm these observations.

It has been reported that exencephaly can also be induced by other teratogenic factors or agents, such as hyperthermia or valproic acid,^58,59 with which a common hierarchy of sensitivity has been observed, possibly because of a common mechanism for all teratogens that cause exencephaly, irrespective of the teratogenic agent.

Chemical interaction studies can often be used to determine whether chemical agents show antagonism, synergism, potentiation, or amelioration in relation to any given effect.⁶⁰ Accordingly, it is known that teratogenicity and fetolethality of cadmium is influenced by simultaneous administration of other chemical agents, such as other metals. For example, zinc almost completely blocks the teratogenicity and fetolethality of Cd,⁶¹ with the effect being antagonic and not synergistic.⁶² It has also been reported that pretreatment with Cd protects the animals to some extent from the teratogenicity of the subsequent administration of a higher dose of the same metal.⁶³ There is also evidence that a subteratogenic dose of caffeine can ameliorate Cd-induced forelimb ectrodactyly in the Cd-sensitive C57BL/6J inbred mouse strain.⁶⁰ In another study, pretreatment with bismuth nitrate ameliorated the teratogenicity of Cd, including exencephaly and abnormalities of the axial skeleton, caused by a single intraperitoneal injection of cadmium sulfate.⁴ Moreover, the beneficial influence of glycine,^64,65 vitamin E, β-carotene, or a combination of the latter two has been demonstrated regarding a reduction in the harmful effects of Cd.⁶⁶

In the current study the co-administration of Spirulina at 500 mg/kg with an injection of CdCl₂ reduced from 59.6% to 1.4% the exencephaly percentage relative to the value produced by administration of Cd alone at 1.5 mg/kg. A dose-dependent protective effect of Spirulina was clearly observed. As reported previously in mice, rats, and hamsters, the administration of Spirulina alone did not produce any alterations in the fetuses, even when using concentrations that represented 10%, 20%, or 30% of the diet.^67
–69 Also, no other toxic effects have been found in relation to alga consumption in general.³⁰

The teratogenicity of many xenobiotics is thought to depend at least in part upon their bioactivation by embryonic cytochromes, prostaglandin H synthase, and lipoxygenases of electrophilic and/or free radical intermediates that covalently bind to or oxidize cellular macromolecules such as DNA, protein, and lipid, resulting in in utero death or teratogenesis.⁷⁰

Specifically, Cd produces oxidative modification of DNA, associated with an increased production of reactive oxygen species,²² while at the same time it decreases the activity of antioxidant enzymes and increases NADPH oxidase activity and lipid peroxidation,^71,72 which contributes to the development of serious degenerative changes in several tissues.⁷³

At 500 mg/kg Spirulina, which gave the best protective effect, the concentration of hydroperoxides in the embryos diminished significantly, reaching the same concentration as the Spirulina group, which was in turn slightly less than the saline control group. This demonstrates that one of the possible mechanisms of the protective action of Spirulina against the teratogenicity of Cd is the direct inhibition of lipid peroxidation and the scavenging of free radicals, which has been found to be true in a variety of diseases (in both humans and animal models) in which oxidative stress is implicated.⁷⁴

This activity might be mostly associated with phycocyanin, a biliprotein found in large concentration in Spirulina, which has been shown to be a powerful antioxidant activity in different experiments probably by increasing antioxidant enzyme activity.^{26,75

–79} Moreover, the chromophore phycocyanobilin, found in Spirulina, also has been found to be a potent inhibitor of NADPH oxidase activity.⁴⁷ Besides, Spirulina also has a significant effect on free radical scavenging,³⁷ an activity that has also been attributed to other SP components.⁴⁴

Studies have shown that the synergistic action of a wide spectrum of antioxidants is more efficient than the activity of a single one. In a finding further confirming the advantages of Spirulina, antioxidants from natural sources (primarily food) have a higher protective efficacy than those of a synthetic origin.⁸⁰

Therefore, we propose that long-term use of Spirulina might help in the prevention of teratogenesis-associated complications. The multifunctional role of Spirulina species potentially makes it an ideal natural drug with immense prophylactic and therapeutic properties.²⁹ However, the extrapolation of these results to humans needs further in-depth study.

Footnotes

Acknowledgments

This work was supported in part by grant 200708555 from SIP, IPN, Mexico. N.P.-C., G.E.-C., R.P.-P., and G.C.-C. are fellows of the EDI and COFAA/IPN Programs.

Author Disclosure Statement

No competing financial interests exist.

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