Potential for genotoxic and reprotoxic effects of vanadium compounds due to occupational and environmental exposures: An article based on a presentation at the 8th International Symposium on Vanadium Chemistry, Biological Chemistry, and Toxicology, Washington DC, August 15–18, 2012

Abstract

Research on the biological effects of vanadium in humans has shown that acute poisoning in workers can manifest itself in a number of symptoms. There are no reports in humans about reproductive and developmental effects induced by vanadium compounds in humans; however, some studies with rats and mice indicate that vanadium can cross the placental barrier and accumulate in fetal membranes rather than the fetus itself. In this case, probably most consequences of administration of vanadium to pregnant females like reabsorptions, fetal death and reduction in size can be the result of maternal toxicity. Concerning genetic and related effects in humans exposed to different vanadium compounds, data are controversial. Data on genotoxic effects in workers exposed to vanadium indicate that they can have an increased risk to develop cancer, and DNA instability can give rise to an onset of genetic syndromes, fetal malformations, and cancer. This paper presents materials presented at the 8th International Symposium on Vanadium Chemistry, Biological Chemistry, and Toxicology in a session titled ‘Relationship between occupational and environmental exposure to vanadium compounds and the reprotoxic and genotoxic effects’.

• Chromosomal aberrations
• comet assay
• fetal malformations
• micronuclei
• teratology
• vanadium pentoxide

Introduction

The use of metals has been a decisive step in cultural and technological development. Several metals perform various, necessary biological functions, while other metals are not required by living organisms. Most metals, however, tend to accumulate in biological systems, and organisms display little adaptability when metals accumulate in high concentrations, causing various alterations.

In unicellular organisms, such disturbances can lead to changes in DNA synthesis and repair, genetic instability, changes in cell proliferation, or cell death. In multicellular organisms (including humans), in addition to the effects mentioned above, the alterations may cause cellular aging, changes in differentiation or cell cycle progression, and the triggering of degenerative processes, which compromise the integrity of cells and tissues. A metal that has acquired special importance for its therapeutic applications and toxicity is vanadium.

Vanadium: Physicochemical properties and biochemical behavior

Vanadium (V) is the first transition metal in Group 5 of the periodic table, followed by niobium (Nb) and tantalum (Ta). Vanadium naturally occurs in low concentrations and, although this metal is essential for many organisms, its requirement in humans has yet to be determined (Rydzynski & Pakulska, 2012). However, the widespread use of vanadium and its release into the environment has led to an increased presence in various ecosystems and in food chains. Thus, humans and other living organisms are in near-constant contact with this metal (Rodríguez-Mercado & Altamirano-Lozano, 2006).

The most common oxidation states for vanadium, which also have well-known biological functions, are III, IV, and V; however, vanadium can exist in lower, very strongly reducing, states. Usually, in biological systems, V(III), V(IV), and V(V) convert from one to another by transfer of an electron; in cells, the metal can most often be found in the form of a metal cation as V³⁺ and as anions/cations as [VO(OH)₃]⁻, [(VO)₂(OH)₅]⁻, or VO²⁺ in the case of V(IV) and , , , or for V(V). In general, VO²⁺ is one of the most stable diatomic molecules (Crans et al., 2004; Hirao, 2000; Rehder, 1991, 2003; Tracey et al., 2007). The inter-conversion of chemical species is complex; for example, V(III) species are unstable in the presence of oxygen and physiological pH, and under physiological conditions, V(IV) species are oxidized to V(V). The latter can easily enter a cell via ion channels, such as those for phosphate and sulfate; this feature renders V(V) very toxic. Furthermore, metabolic processes can transform V(V) to V(IV) inside the cell (Rehder, 2003; Tracey et al., 2007).

More detailed information about other sources of exposure are described in other articles from the 8th International Symposium on Vanadium Chemistry and Toxicology (August 2012). The remainder of this paper only describes effects of vanadium agents on genetic material and reproduction.

Vanadium genotoxicity

Human studies

Although humans are exposed to vanadium compounds either occupationally by inhalation or via ingestion of food, few epidemiological studies have been designed to evaluate DNA damage in human populations (IARC, 2006). In one of the few human studies, Ivancsits et al. (2002) evaluated the genotoxic effects of exposure to V₂O₅. Forty-nine workers at a factory producing this metal participated in the study, and it was determined that the V concentrations in the workers’ serum (7.73 μg V/L) and urine (14.57 μg V/g creatinine) were higher than in the control group, 3.43 μg/L and 1.13 μg V/g creatinine, respectively. However, when researchers assessed DNA damage in blood cells using a sister chromatid exchange (SCE) assay and single-cell gel electrophoresis (Comet assay) under alkaline conditions, they did not observe any increase in chromatid exchanges or DNA migration, concluding that vanadium does not cause DNA damage in vivo.

Similarly, in another epidemiological study involving 52 workers who were exposed to V₂O₅, the metal concentration in plasma was 2.2 μg V/L, which was higher than in the control group (0.3 μg V/L). In addition, when leukocytes from whole blood were subjected to the Comet assay, no increase in DNA damage was observed. However, testing of micronuclei (MN) in lymphocytes with cytokinesis blockade yielded an observable increase in the number of micronucleated cells and in the percentage of necrotic cells. Therefore, it was concluded that occupational exposure might increase the risk of contracting certain chronic degenerative diseases and that protective measures for workers should, thus, be improved (Ehrlich et al., 2008).

Animal studies

Animal studies have demonstrated that the accumulation of vanadium in the body occurs in the bone, kidney, liver, spleen, testes, intestines, and stomach, and the intracellular distribution in these tissues indicates that the nucleus is the organelle that retains the highest amount of vanadium, which can thus interact with DNA (Sabbioni & Marafante, 1978; Sabbioni et al., 1991; Villani et al., 2007). The acute toxicity of vanadium compounds is low when administered orally, moderate when inhaled, and high when injected intraperitoneally (IP). In addition, these studies indicate that small animals such as rats and mice tolerate these compounds better than do larger animals such as rabbits and horses (IARC, 2006).

The administration of sodium orthovanadate (Na₃VO₄), ammonium metavanadate (NH₄VO₃), and vanadyl sulfate (SVO₅), at doses of 75, 50, or 100 mg/kg, respectively, to CD-1 strain male mice intragastrically induced an increase in MN frequency in bone marrow cells evaluated during a 72-h period. Simultaneously, a second experiment conducted for 24 and 36 h determined that only SVO₅ caused structural chromosomal aberrations (CAs), but that all the compounds induce numerical CA (hypo-, hyper-, and polyploid cells), thus confirming that vanadium exerts more aneuploidogenic effects than clastogenic effects in vivo (Ciranni et al., 1995).

This effect was also observed in germ cells when female ICR mice were treated IP once with 5, 15, or 25 mg of Na₃VO₄/kg and subsequently (i.e. 18 h later) evaluated for cytogenetic oocyte abnormalities and for frequencies of hypo-, hyper-, hap-, di-, eu-, and tetraploid cells in their bone marrow (Mailhes et al., 2003). The marrow samples from the treated hosts exhibited increased numbers of tetra- and hyperploid cells, and premature centromere separations, whereas oocytes displayed an increased number of early anaphase cells. These alterations, in turn, led to poor segregation of chromosomes during subsequent cell divisions.

In another study, male 102/E1 x C3H/E1 mice were treated IP with Na₃VO₄ in doses similar to those of the previous investigation. The effect on cellular proliferation in germ cells was evaluated, and the frequency of hyperploidy and diploidy was determined by fluorescence in situ hybridization (FISH). By specifically marking chromosomes X, Y, and 8, it was observed that the vanadium compound did not induce cell cycle delay but increased the number of hyper-haploids after treatment with the highest doses (i.e. 15 and 25 mg/kg). YY8 and XX8 were the most common aneuploidies in the sex chromosomes, indicating that poor segregation of chromosomes occurs during the second meiotic division by non-disjunction of sister chromatids (Attia et al., 2005).

Furthermore, induction of genotoxic and cytotoxic effects on bone marrow cells has not been demonstrated by the MN assay. In these two studies, it was demonstrated that Na₃VO₄ does not arrest meiotic divisions completely during either male or female meiosis. It was also proposed that male germ cells are more sensitive than somatic cells to the aneugenic effects of orthovanadate (Attia et al., 2005; Mailhes et al., 2003).

Similarly, the ability of V₂O₅ to induce primary DNA lesions, such as single-strand breaks, in testicular cells from male CD-1 mice was evaluated by a Comet assay 24 h after IP administration of 5.75, 11.50, or 23 mg of V₂O₅/kg. The data revealed that this compound has a dose-dependent genotoxic effect (Altamirano-Lozano et al., 1996). Using the same protocol, Altamirano-Lozano et al. (1999) evaluated this function of V₂O₅ in liver, kidney, lung, spleen, heart, and bone marrow cells, noting that treatment induced DNA damage in all organs, with the liver, kidneys, and heart being most susceptible. The investigation also revealed bone marrow cells are less sensitive to damage induced by vanadium (as noted in other studies), indicating that dividing cells can repair this type of damage better than cells with low proliferation rates can.

Studies have been conducted using CD-1 male mice administered Na₃VO₄ orally in drinking water for 5 weeks at concentrations of 7.5, 75, 750, or 1500 mg/L. Genetic damage was evaluated in various tissues employing MN assays in bone marrow cells and peripheral blood reticulocytes. Primary lesions were evaluated by the Comet assay in splenocytes, testicular cells, and bone marrow; the sperm chromatin structure was also analyzed. The results revealed no significant damage in the germ cells, indicating that oral exposure to vanadium is not genotoxic in these cells; however, damage was observed in the somatic cells only at the highest doses, possibly because of the low bioavailability of vanadium in these cells (Leopardi et al., 2005).

Villani et al. (2007) evaluated the effects of oral administration of VOSO₄ on male CD-1 mice at doses of 10, 100, 500, or 1000 mg/L for 5 weeks, using the Comet assay and MN test, which yielded no treatment-related changes; thus, this metal (as V[IV]) was not genotoxic in somatic and germ cells. However, given the various biological effects on cellular mechanisms, the authors recommend caution in applying these results to the evaluation of other vanadium compounds.

Vanadium reproductive toxicity

Human, animal, and in vitro studies suggest that heavy metals may exert adverse effects on the reproductive health of males, even at relatively low concentrations. Heavy metals can affect the male reproductive system, altering the hypothalamic-pituitary-gonadal axis or directly affecting spermatogenesis, thus diminishing the semen quality (Leopardi et al., 2005; Mendiola et al., 2011). Of the toxic metals, arsenic, cadmium, chromium, lead, mercury, and vanadium have been identified as highly significant in environmental and occupational exposures, as studies have shown that these metals can accumulate in the testis and/or epididymis, altering reproductive and endocrine function (Castellini et al., 2009; Clarkson et al., 1985; Danielsson et al., 1984; Thompson & Bannigan, 2008).

As with many metal agents, effects of vanadium on reproduction depend on several factors, such as the chemical form of the compound, state or oxidation number of vanadium in the compound, route of exposure, duration of administration, and dosage (Domingo, 1996) (Table 1). Information on reproductive toxicity caused by vanadium compounds is limited and is non-existent in humans; however, interest in vanadium coordination compounds (e.g. oxovanadates) has increased in recent years due to the pharmacological properties of the compounds, which can be employed for treating diabetes mellitus or controlling cholesterol levels. Other areas of increased attention are the effects of these coordination complexes in cancer treatment and their spermicidal activities (Less et al., 2006). However, a serious caveat in the latter application is suggested by the laboratory animal data revealing that vanadium can permanently damage reproductive function.

Table 1. Developmental and maternal toxicity produced by different vanadium compounds (studies listed chronologically).

Compound	Concentration	Period of treatment	Species	Route	Maternal effects	Embryo and fetal effects	References
Ammonium metavanadate	0.47, 1.88, 3.75 mg/kg/day	5–10 d of gestation	Hamster	Intraperitoneally	No significant effects	Skeletal anomalies	Carlton et al. (1982)
Vanadium pentoxide	0.9 single dose	3 or 8 d of gestation	Mice	Intravenous	No effects reported	Skeletal ossification decreased (day 8). Broken spinal cord (9% of fetuses)	Wide (1984)
Sodium metavanadate	5, 10, 20 mg/kg/day	Males: 60 d before mating. Females: 14 d before mating; through gestation and lactation	Rat	Drinking water	No adverse effects	Significant decreases in development	Domingo et al. (1986)
Sodium metavanadate	5, 10, 20 mg/kg/day	6–14 d of gestation	Rat	Drinking water	No significant adverse effects	No significant adverse effects	Paternain et al. (1987)
Vanadyl sulfatepentahydrate	37.5, 75, 150 mg/kg/day	6–15 d of gestation	Mice	Drinking water	Decreased liver, kidney weight	Decreased fetal weight, increased embryo lethality, skeletal defects	Paternain et al. (1990)
Sodium orthovanadate	7.5, 15, 30 mg/kg	6–15 d of gestation	Mice	Drinking water	Reduced weight gain and food consumption. Death in 30 mg.	Delayed skeletal ossification in some areas	Sanchez et al. (1991)
Sodium metavanadate	2, 4, 8 mg/kg	6–15 d of gestation	Mice	Intraperitoneally	Decreased weight gain	Reduced weight, increased embryo lethality, cleft palate at high dose	Gómez et al. (1992)
Vanadium pentoxide	8.5 mg/kg	6–15 d of gestation	Mice	Intraperitoneally	No maternal toxicity observed.	Both proportion of litters with abnormal fetuses and number of altered fetuses was higher. Limb shortening most frequent alteration	Altamirano-Lozano et al. (1993)
Sodium metavanadate	0.25, 0.50 mg/ml	Gestation	Normal and diabetic female rats	Drinking water	Vanadate treatment reduced both conception rate and ability to carry pregnancy to term; effects more severe in diabetic versus non-diabetic dams	Not reported	Ganguli et al. (1994)
Fine particulate matter (PM2.5)	Oil-combustion associated elements vanadium and nickel	Exposure throughout pregnancy	Pregnant women	Inhaled	Not reported	Increased risk of low birth weight	Bell et al. (2010)

Effect in males

Epidemiological studies and occupational exposure studies have reported that certain metals exert direct effects on sperm cells, reducing their motility and/or affecting their morphology (Kumar, 2004; Xu et al., 1993). In addition, animal studies and in vitro studies have, in most cases, demonstrated a positive correlation between effects on sperm and the metal concentration in semen (Aragón & Altamirano-Lozano, 2001; Castellini et al., 2009; Sakhaee et al., 2012) (Table 2).

Table 2. Reprotoxicity induced in males by vanadium compounds (studies listed chronologically).

Compound	Concentrations	Dose and period	Species	Route	Effects	References
Vanadium sulfate	0.08 mM/kg	Single dose	Rats	Intratesticular	Reduction in testis weight and total necrosis	Kamboj & Kar (1964)
Sodium metavanadate	5, 10, 20 mg/kg/day	60 d before mating	Rats	Orally	No effects on fertility and reproduction	Domingo et al. (1986)
Vanadium pentoxide	8.5 mg/kg bw	Every 3 d for 60 d	CD-1 male mice	intraperitoneally	Decrease in fertility rate. Sperm count, motility, and morphology were impaired with the advancement of treatment. Decreased implantation, live fetuses, and fetal weight and increased the number of resorption/dam.	Altamirano-Lozano et al. (1996)
Vanadium pentoxide	12.5 mg/kg	Male: every second day, from birth to 21 d. Female: from day 21 to day of first vaginal estrus	Pre-pubertal rats	Intraperitoneally	Males: increased weight of seminal vesicles, thymus and submandibular glands Females: no significant differences in age of vaginal opening nor first vaginal estrus. Ovulation rate was lower in treated animals	Altamirano et al. (1991)
Sodium metavanadate	20, 40, 60, 80 mg/kg/day	64 d before mating	Male mice	Drinking water	Reduced body and epididymis weight. Decrease in sperm count	Llobet et al. (1993)
Vanadium tetroxide	4.7, 9.4, 18.8 mg/kg	60 d	Male mice	Intraperitoneally	Increase of apoptosis and ultrastructural changes of germ cell, but testosterone level was not modified	Aragón et al. (2005)
Sodium orthovanadate	0.75–1500 mg/l	5 weeks	Male CD-1 mice	Drinking water	No treatment-related effect on sperm chromatin structure or on testis cell population was observed	Leopardi et al. (2005)
Sodium orthovanadate	5, 15, 25 mg/kg	Sperm were sampled from epididymis 22 d after IP injection of host	Male mice	Intraperitoneally	Significant increases in the frequencies of total hyper-haploid sperm were found with 15 and 25 mg/kg, indicating induced non-disjunction during male meiosis	Attia et al. (2005)
Vanadium pentoxide	0.02 M	1 h/twice a week, 12 weeks	Male mice	Inhaled	Vanadium accumulates in the testes and decreases the percentage of gamma-tubulin in testicular cells (Sertoli, Leydig, and germ cells)	Mussali-Galante et al. (2005)
Sodium metavanadate	0.4 mg V/kg	Daily for 26 d	Male Sprague Dawley rats	Intraperitoneally	Reduced sperm count associated with decreased serum testosterone and gonadotropins level	Chandra et al. (2007a)
Vanadyl sulfate	100 mg/kg/day	60 d	Male Wistar rats	Orally	Decreased weights of testes and accessory reproductive organs. Reduced number and motility of epididymal sperm. Mating tests with untreated females revealed a decrease in pregnancy rate and mean number of pups delivered	Jain et al. (2007)
Sodium metavanadate	1, 10, or 100 µM	4 or 6 h	Ejaculated sperm samples, New Zealand White rabbits	In vitro	Reduced sperm motility and curvilinear velocity and numerous folds in the acrosome membrane	Castellini et al. (2009)

In vitro studies

In many in vitro studies, Leydig cells and germ cells have been identified as the main targets of cytotoxicity, reducing steroidogenesis, and disrupting spermatogenesis (Laskey & Phelps, 1991). Furthermore, metals can affect the junction between Sertoli cells, altering the viability of sperm in the epididymis. The exact cause is unknown, but proposed mechanisms of metal toxicity include oxidative stress, inflammation, induced apoptosis, and ionic or molecular mimicry. Vanadium produces reactive oxygen species (ROS), causing structural lipid peroxidation and alteration of the anti-oxidant activity of some enzymes, mainly superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) (Aragón et al., 2005; Byczkowski & Kulkarni, 1998; Marouane et al., 2011).

Although exact mechanisms by which vanadium compounds immobilize sperm are unknown, human spermatozoa are sensitive to oxidative stress because of the high content of polyunsaturated fatty acids in their cell membrane. These cells also have low levels of cytoplasmic enzymes to trap the ROS, which causes lipid peroxidation and reduces the activity of enzymes that repair oxidative damage. D’Cruz et al. (1999) reported that several coordination complexes containing V(IV) (vanadocenes) exhibit potent activities in human spermatozoa as spermicides. The authors found that the spermicidal activity exhibited by these V(IV) coordination complexes is related to their ability to catalyze ROS generation and to cause apoptosis.

In vivo studies

When administered intratesticularly to male rats, V(IV) compounds caused necrosis and a reduction in gonadal weight (Kamboj & Kar, 1964). In addition, these compounds reduced the number of sperm cells, decreased their motility, and caused increased frequency of morphological abnormalities while modifying the serum concentrations of hormones such as testosterone, LH, and FSH (Chandra et al., 2007a). Histopathological examination revealed an inhibition of spermatogenesis; in addition these authors postulated that increased formation of free radicals during exposure to vanadium could render the testicle more susceptible to oxidative damage (Chandra et al., 2007b).

Altamirano-Lozano et al. (1996) studied male CD-1 mice treated IP with 8.5 mg V₂O₅/g every 3 d for a period of 60 d; sub-groups of mice were then euthanized every 10 d for analyses. An additional group was mated with untreated females 24 h after the final V₂O₅ dose. The results showed that treatment with vanadium decreased overall fertility, number of fetal implants, number of live fetuses, and fetal weight, and increased the incidence of resorptions. Decreased numbers and decreased motility of spermatozoa, along with an increased frequency of abnormal sperm morphology after treatments, were also noted. DNA damage in testicular cells was also evident. In a study of mice exposed to inhaled V₂O₅ (at 0.02 M V₂O₅) twice a week for 12 weeks, it was found that the compound accumulated in the testes within 24 h of host exposure and generally reduced the percentage of α-tubulin in all analyzed testicular cells, altering microtubules, impairing cell division, and disrupting spermatogenesis (Mussali-Galante et al., 2005).

Vanadium tetroxide (V₂O₄), when orally administered to male CD-1 mice in daily dosages of 9.4 and 18.8 mg/kg for 60 d, induced degenerative changes in the seminiferous tubules and reduced testosterone levels. In this case, testicular weight remained unaffected; however, sperm motility and viability were decreased, changes in the morphology of spermatozoa were present, and degenerating germ cells in the seminiferous epithelium and increased numbers of apoptotic cells were observed. The concentration of progesterone and testosterone was not affected by treatment (Aragón et al., 2005).

Jain et al. (2007) orally administered 100 mg vanadyl sulfate (VOSO₄)/kg to male rats daily for 60 d, and decreases in weight of the testes and accessory sex organs, numbers and motility of spermatozoa, and atrophy and/or reduced seminiferous tubule diameters were determined. At the histopathological level, the researchers noted that nuclei of Leydig cells were reduced in size. Furthermore, tissue analysis revealed an altered biochemical environment in the genitals. Five days prior to the end of treatment, each male was placed in the same box with two untreated females. The fertility rate was determined to be decreased along with the number of offspring per litter. In this study, the authors propose that the reduced weight of the testis may have been due to the decreased number of germ cells, and the reduced number of cells may have been attributable to a failure in the maturation of sperm and disruption of the secretory function of the epididymal cells, possibly caused by androgen insufficiency. Alternatively, all of the above-mentioned findings might have been caused by oxidative stress or by failure to generate energy for metabolic enzymes.

D’Cruz & Uckun (2000) evaluated four different vanadocene compounds in concentrations of 7.5 mg/kg for 28 d by intra-testicular administration and found decreased weight and number of spermatozoa in the epididymis, atrophy and almost complete vacuolation of the seminiferous tubules, and loss of late spermatids. These compounds produced germ cell apoptosis.

Maternal toxicity and effects of vanadium on embryo and fetus

Animal studies

Vanadium in the form of vanadate is a potent inhibitor of tyrosine phosphatase inhibitors, including CDC25, in human kidney cells. ATPase activity is necessary for the assembly and activation of the mitotic apparatus and spindle fibers. In Xenopus and mouse oocytes, vanadate decreases the activity of the maturation-promoting factor through inhibition of p34 cdc2 tyrosine dephosphorylation. Apparently, vanadate also inactivates the maturation factor of porcine oocytes (i.e. vanadate potentially inhibits or disturbs oocyte maturation and correct assembly of the mitotic spindle). This effect compromises the segregation of oocyte chromosomes and damages their descendants (Kim et al., 1999).

Mailhes et al. (2003) found that vanadate administered intraperitoneally immediately after application of hCG, increased the occurrence of premature anaphase in oocytes, while, in bone marrow cells, vanadate also increased the percentage of tetraploids, hyperpolyploids, and premature centromere separation in bone marrow cells. The researchers postulated that a kinase-phosphatase imbalance during oocyte maturation and the metaphase-anaphase transition produce cytogenetic abnormalities that differ between oocytes and bone marrow cells.

The embryos of all mammals depend on the mother’s system; thus, the environment in which the mother lives is of great importance in successful development of the offspring. Naturally, the age and physical and nutritional status of the mother could exert significant effects during growth in the uterus; however, if integrity of the maternal system is altered, the change may indirectly affect the offspring. Studies of intrauterine development have revealed that exposure to physical and chemical agents, infectious diseases, hormonal changes, and nutritional deficiencies or excesses are factors that directly cause abnormal embryonic or fetal development and birth defects (Scialli, 1992).

While there is little information on the reproductive toxicity of vanadium and developmental toxicity after vanadium inhalational exposure, this metal causes toxicity in the embryo and fetus when administered orally. However, the toxic effects of vanadate and vanadyl have been observed only at doses remarkably higher than the doses that can be ingested through food (Domingo, 1996). Vanadium can cross the placental barrier (Edel & Sabbioni, 1989; Paternain et al., 1990) but appears to accumulate in fetal membranes rather than in the fetus itself (Roshchin et al., 1980; Hackett & Kelman, 1983; IPCS, 1988). However, Underwood (1977) determined that the metal could accumulate in the fetal skeleton.

Roshchin et al. (1980) reported that administration of vanadium (oxidation state undefined) to pregnant rats on days 21 and 22 of gestation causes accumulation of this metal in the placenta; however, vanadium does not cross the placental barrier. Nonetheless, the researchers observed some embryotoxic effects, like increased mortality, when vanadium was administered on day 10 of pregnancy. Wide (1984) found that intravenous administration of 0.15 ml of 1 mM V₂O₅ (V⁵⁺) to mice on day 8 of gestation caused a decrease in skeletal ossification areas in 71% of fetuses, with an increased number of non-viable implants, and 9% of fetuses examined on day 17 of gestation had a broken spine.

In hamsters, a mild transplacental effect was reported after applying ammonium metavanadate (V⁵⁺, 0.47, 1.88, and 3.75 mg NH₄VO₃/kg/day) intraperitoneally to pregnant females from days 5–10 of gestation. No toxic effects were observed in the mother at any dose, but skeletal abnormalities occurred, e.g. micrognathia, supernumerary ribs, and alterations in ossification of the sternebrae. Despite these abnormalities, it was suggested that the low incidence and lack of a dose-dependent response was not definitive proof that NH₄VO₃ was teratogenic (Carlton et al., 1982).

Edel & Sabbioni (1989), after applying intravenous injections of pentavanadate with [⁴⁸V]-labeled vanadium at doses of 0.1 μg V⁵⁺/animal to pregnant rats on day 12 of gestation, found significant amounts of vanadium in the liver, intestine, and kidney of fetuses, proving that vanadium in this form crosses the placental barrier and is metabolized by the fetus. Moreover, V⁴⁺ crosses the placenta and reaches the fetus, as demonstrated by the results reported by the same authors when applying vanadyl sulfate pentahydrate (V⁴⁺) to pregnant female mice by intragastric gavage on days 6–15 of pregnancy. In this case, the authors postulated that the transport path through the placenta may be facilitated by the formation of a complex with transferrin or albumin.

Altamirano et al. (1991) evaluated the effect of V₂O₅ on the early reproductive process in newborn rats. The researchers intraperitoneally injected pre-pubertal male and female rats of the CII-ZV strain every 2 d (from birth until day 21) at a dose of 12.5 mg/kg and another group of females from day 21 until the day of the first vaginal estrus. In the pre-pubertal and juvenile females, no differences were observed in the vaginal opening or estrous cycle; however, the ovulation rate was decreased in young females treated with this compound. Notably, when juvenile females were treated from day 21 post-partum, there was an increase in weight of the submandibular glands, thymus, and liver. In the males, an increase was also detected in the seminal vesicles, thymus, and submandibular glands. These results demonstrate that, similar to other metals, the toxicology of vanadium has sex-based differences, with the male pre-pubertal group being more susceptible than the female group.

As with the majority of toxic agents, the path or route of exposure is critical to the effect produced, and this phenomenon is clearly observed in the case of vanadium compounds. The oral administration of sodium metavanadate (20 mg NaVO₃/kg/day) to rats on days 6–14 of gestation does not cause embryolethal or teratogenic effects. However, if the NaVO₃ is administered via the intraperitoneal route to female mice from days 6–15 of gestation at doses of 4 or 8 mg/kg/day, then an increase occurs in the number of resorptions, dead fetuses, and cleft palates (Gomez et al., 1992). Although the authors of this study reported the presence of maternal toxicity, they postulated that the effect observed in fetuses may be caused by direct contact of vanadium with the tissues from embryos or fetuses and not by maternal toxicity; however, it is likely that most of the effects produced by the administration of vanadium compounds to pregnant females (increased percentage of resorptions, fetal death, and fetal weight reduction) is the result of maternal toxicity caused by high doses of the compounds (Léonard & Gerber, 1998).

The oxidation number of vanadium in compounds is also a key factor explaining the different responses found during assessments of developmental toxicology. For example, intra-gastric administration of sodium orthovanadate (V⁵⁺; at 0, 7.5, 15, 30, or 60 mg/kg) to mice on days 6–15 of gestation caused maternal toxicity observed as decreased weight and even death; however, no teratogenic effects or embryo lethality were observed, although delayed ossification occurred only at the highest dose (Sánchez et al., 1991). Furthermore, oral administration of vanadyl sulfate pentahydrate (V⁴⁺; up to 150 mg/kg/day) to female mice from days 6–15 of gestation caused maternal, embryonic, and fetal toxicity (including teratogenicity) at all doses—especially the highest dose—tested, with cleft palate and micrognathia being the major malformations observed (Paternain et al., 1990).

Regarding sodium metavanadate (NaVO₃), in the literature, there was only one study reporting a significant increase in number of reabsorbed fetuses (Paternain et al., 1987). In a subsequent study with V₂O₅ by Altamirano-Lozano et al. (1993), among female CD-1 mice that were injected daily from days 6–15 of gestation with 8.5 mg V₂O₅/kg IP, vanadium did not cause a change in the numbers of live versus dead fetuses or the number of implants; however, it did lead to decreases in fetal weights at day 18 of gestation and caused significant delays in skeletal ossification (i.e. there were decreased numbers of ossification centers in extremities). No maternal toxicity was noted at this dose; nevertheless, the authors concluded that vanadium altered the fetal development process.

In contrast, oral administration of V₂O₅ (V⁵⁺) to weanling rats (10–200 μmol/kg) for 3 d resulted in increased alkaline phosphatase activity and DNA content in the femoral diaphysis of treated animals. These data indicate that vanadium plays a role in bone formation (Yamaguchi et al., 1989), a finding consistent with reports of vanadium affinity for bone in developing animals.

Effects of vanadium on reproduction and fetal development in diabetics

Human studies

One use of vanadium is diabetes treatment, as certain compounds have proven to be insulin-mimetic, helping to control the disease. However, there is limited data in humans, although research performed on laboratory animals has yielded significant results. Manci et al. (1989) and Al-Attas et al. (1995), who analyzed placentas of diabetic and non-diabetic women, measured lower vanadium levels (as vanadate compounds) in the case with gestational diabetes mellitus (7.62 μg V/g) compared with the control (8.73 μg V/g), which was attributable to decreased intake or absorption and increased utilization or excretion of vanadium. The researchers proposed that the binding of vanadium to the maternal tissue in humans is increased in diabetes mellitus when insulin is deficient. Under this hypothesis, recent studies in vitro have revealed that the binding of vanadate to insulin receptors increases in the placenta of women with gestational diabetes mellitus.

Animal studies

Given the interest in the possible use of vanadium to treat diabetes during pregnancy, the effect of oral administration of vanadate on the reproductive process was evaluated in normal and diabetic rats. Vanadate was administered in the drinking water of animals at concentrations of 0.25 and 0.50 mg/ml, and the data revealed that vanadium did not normalize blood glucose levels in diabetic pregnant rats; however, the metal was found to alter certain reproductive parameters, such as the pregnancy rate and the ability to sustain the pregnancy to term in normal and diabetic rats. The authors postulated that these results were most likely due to changes in the estrous cycle, as polycystic ovaries were observed in the females that did not become pregnant. Post-mortem analysis of the females that did not reach term revealed highly vascular and fluid-filled uteri, indicating that these animals had conceived and perhaps had experienced early, spontaneous abortions (Ganguli et al., 1994).

In another study, normal and diabetic rats received 0.25 mg NaVO₃/ml in drinking water during gestation. The presence of vanadium in maternal blood had a negative effect on fetal development, namely, a marked reduction in the number of live fetuses per litter in both diabetic animals and normal animals. This toxicity may have been caused by a transplacental transfer of a significant amount of vanadate from the maternal to fetal compartments. Alternatively, the negative effect of vanadium in the blood of developing fetuses might be a consequence of the excessive generation of free radicals and reactive species, as it has been documented that high levels of reactive species can result in embryonic death (Eriksson & Borg, 1991). Based on these above-noted results, oral treatment with vanadate is toxic and ineffective for gestational diabetes because it reduces the reproductive capacity and interferes with fetal growth and development in normal animals and diabetic animals. Therefore, vanadium treatment may be contraindicated for diabetic females of reproductive age (Ganguli et al., 1994).

Conclusion

Metals can operate through hormonal or genotoxic pathways, and some metals can penetrate the blood–testis barrier, affecting spermatogenesis by altering the integrity of the genetic material, altering hormone production, or affecting the cell cycle in certain cases (e.g. during meiotic non-disjunction). This damage can cause adverse effects in the offspring. At high doses, vanadium compounds can damage a developing organism in the uterus, but, apparently, this effect is mainly due to maternal toxicity. Because vanadium salts are inefficiently transferred to the fetus itself, fetal malformations are found only at very high doses (specific agents and corresponding doses reviewed in Léonard & Gerber, 1998). The available data indicate the necessity for more studies of the effects of vanadium in occupationally-, environmentally-, or pharmacologically-exposed human populations. As demonstrated in the present review, animal models have been shown to indicate that the reproductive toxicity of various vanadium compounds depends on the dose, duration of treatment, route of administration, sex, and species that is encountered.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.