Effects of maternal vanadate treatment on fetal development

Life Sciences, Vol. 55, No. 16, pp. 1267-1276, 1994 Copyright © 1994 Elsevier Science Ltd Printed in the USA. All rights reserved 0024-3205/94 $6.00 + .00

Pergamon 0024-3205(94)00267-3

EFFECTS OF MATERNAL VANADATE TREATMENT ON FETAL DEVELOPMENT Supriya Ganguli, Donald J. Reuland, LeRoy A. Franklin, Donald D. Deakins, William J. Johnston, and Asiya Pasha Indiana University School of Medicine Terre Haute Center for Medical Education and Indiana State Universtiy Department of Chemistry Terre Haute, IN 47809 (Received in final form August 3, 1994) Summary. Oral vanadate treatment is effective in normalizing blood glucose in both Type 1 and Type II diabetics. Using Sprague Dawley rats we examined the effectiveness of such treatment in amelioration of hyperglycemia in diabetic pregnancy and its effect on fetal growth in both normal and diabetic pregnant dams. Initiation of vanadate treatment to diabetic and normal pregnant dams increased blood vanadium levels in both groups, but this concentration in the diabetic pregnant group reached approximately twice the value present in the normal group. Despite this high blood vanadium level in the diabetic pregnant dams, oral vanadate treatment was not effective in normalizing blood sugar in this group. Additionally, vanadate treatment was found to be toxic during diabetic pregnancy, causing death to 45% of the test animals. Maternal blood vanadium had a negative effect on fetal development, markedly reducing the number of live fetuses per pregnancy. In summary, oral vanadate treatment is toxic and ineffective during diabetic pregnancies and interferes with fetal growth and development in both normal and diabetic pregnancy. Key Words: vanadate, fetal development, diabetes, diabetic pregnancy

Oxovanadiums are industrial pollutants (1). Among the oxovanadiums, vanadate (VO43) and vanadyl (VO 2+) ions are the most active in biological systems (2), and can produce toxic effects when present in high concentrations (2,3). Vanadate is a trace element in mammalian nutrition and demonstrates various biological effects both under in vivo and in vitro conditions (1). Most of the blood vanadium is present as vanadate or vanadyl ions (1,4).

Corresponding Author:.

S. Ganguli, Ph.D., Indiana School of Medicine, Terre Haute Center, 135 Holmstedt Hall, Indiana State University, Terre Haute, IN 47809

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Recently, oral vanadate treatment has been found to be effective in ameliorating hyperglycemia (5-8) and restoring normal levels of insulin-dependent enzymes involved in the insulin-regulated metabolism of nutrients in both chemically induced and genetically diabetic animal models (1,814). These insulin-dependent enzymes are responsible for stimulating hexose transport, glucose oxidation (8-11), lipogenesis (12,13), and glycogen synthesis (14). Like insulin, vanadate also corrects excess glucose production in diabetic animals by reducing the level of phosphoenolpyruvate carboxykinase (PEPCK), the key gluconeogenic enzyme, at the transcription level (15). The specific mechanism(s) at the molecular level that is responsible for the insulin-like effects of vanadate in the restoration of key insulin-dependent metabolic enzymes, under both in v i v o a n d in v i t r o conditions, is not known. Peroxides of vanadium (IV), peroxovanadates, the product of biologically active oxovanadiums and H202, demonstrate more potent insulino-mimetic properties (2). Aside from their insulinlike actions, vanadate and other oxovanadiums demonstrate a wide range of effects upon biological systems (1,16). Vanadate is a potent inhibitor of key metabolic enzymes, such as Na+,K+-ATPase (1,17), Ca++-ATPase (1,18), protein phosphatases (1,19), acid and alkaline phosphatases, and ribonuclease Vanadate and vanadyl ions can affect the intracellular message transduction axes for many growth factors and cytokines through their modulatory effects on key intracellular events, such as cAMP generation (20,21), protein tyrosine phosphorylation (22,23) and the production of various free radical species (24-27). Vanadate demonstrates a wide range of effects on cell proliferation and differentiation that varies with the vanadate dose and cell type (28-30). Vanadate also exhibits a wide range of toxic effects in biological systems (31-33). The potential for vanadate toxicity is enhanced due to its tendency to accumulate selectively in some tissues, such as the kidneys and heart (34). Vanadate at higher levels can produce sustained vasoconstriction that may impede organ function and inflict tissue damage (35). The specific underlying cause(s) for various beneficial and/or toxic effects of vanadate on cell and organ tissues and metabolism is not clear. A single bolus IP injection of vanadate (25 mg) to pregnant mice at midpregnancy was found to cause mcreased abortions and abnormal fetal growth (36). Chronic oral vanadate treatment of pregnant mice has been shown to increase the loss of conceptus (37). In our laboratory, initiation of chronic oral vanadate treatments (0.25 mg/mL and 0.50 mg/mL of Na~VO4 in 1/2 N saline as drinking water) to normal and diabetic rats prior to breeding caused a significant dose-dependent reduction in their reproductive efficiency and their ability to carry pregnancy to term (accepted for publication). In the present study, we have examined the effects of the late initiation of oral vanadate treatment of pregnant dams on the management of blood glucose and on the development of the fetus during the second half of pregnancy, the period of rapid phase of fetal growth and development in both normal and diabetic rats. Methods

Sprague-Dawley rats were obtained from Harden Laboratories, Indianapolis, IN. The streptozotocin, sodium chloride, sodium orthovanadate and sodium citrate were obtained from Sigma Chemical Co., St. Louis, MO. The insulin (Lente 100 units/mL, Eli Lilly Co., Indianapolis, IN) was obtained from a pharmacy. The kits for glucose analysis (glucose oxidase) were obtained from Sigma Chemical Co., St. Louis, MO. The Keto-Diastix for urine glucose determinations came from Ames Co. Standard rat chow (24.0% crude protein, 6.0% crude fat, 4.5% crude fiber and the remainder, carbohydrates) was from Purina Co., St. Louis, MO. The standard vanadium solution (1 mg/mL) and the graphite furnace tubes were obtained

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from Varian, Sunnyvale, CA. The graphite furnace atomic absorption spectrometer was a Varian SpectrAA-10 with a GTA-96 furnace complete with a programmable sample dispenser.

Experimental Design To determine specifically the effects of maternal blood vanadate levels on the developing fetus from both normal and diabetic pregnant dams without prior exposure to vanadate, we utilized the following experimental model. A total of 30, young, virgin rats weighing 210-230 g were divided randomly into two unequal groups, consisting of 18 and 12 animals. The rats were housed in large cages, 4 animals per cage, at an ambient temperature of 22" C with a 12-hour light/dark cycle. Following one week of acclimatization, all animals in the 18-animal group were injected with streptozotocin, 35 mg/kg, through a tail vein. Streptozotocin was diluted immediately prior to injection in a 50 mM sodium citrate buffer, pH 4.7. All streptozotocin-treated animals were monitored daily for urine glucose, using KetoDiastix. Animals demonstrating urine glucose levels exceeding 1000 mg/dL for four consecutive days were classified as diabetic. One of the 18 animals demonstrated only a trace amount of glucose in the urine following streptozotocin treatment and was eliminated from the experimental group. Confirmed diabetic dams were placed immediately on a daily insulin (Lente, Eli Lilly Co) injection schedule (1-4 U/kg), as needed to maintain urine glucose levels between trace and 500 mg/dL. The diabetic and nondiabetic animals were divided further into two groups each. The 17 confirmed diabetic animals were divided randomly into two unequal groups of 11 and 6 animals. Similarly, the nondiabetic group of 12 female rats also was divided into 2 groups, 7 and 5, respectively. All dams were placed individually with an experienced male in separate cages and their vaginal smears were examined daily for sperm. The appearance of sperm in the vaginal smear was taken as an indicator of successful impregnation, and the time was recorded as day "0" of pregnancy. Impregnated females were removed from males and were housed individually in separate cages. All animals had free access to drinking water and food (rat chow). Normal and diabetic pregnant rats were allowed to advance to day 10 of pregnancy. For the diabetic dams, daily SQ injections of insulin, 1-4 U/kg of body weight, continued through the first 10 days of pregnancy to maintain low urine glucose. On day 10 of pregnancy, the supplemental insulin injections for diabetic animals were suspended, and both nondiabetic (normal) and diabetic pregnant animals were placed into two different treatment groups: no treatment (control) and vanadate treatment groups, as described below. Five nondiabetic (control) and 6 diabetic (diabetic control) animals received 1/2 normal saline without vanadate as drinking water. The diabetic group consisting of 11 animals and the nondiabetic group of 7 animals received vanadate treatment, 0.25 mg/mL sodium orthovanadate, in I/2 normal saline as drinking water. The drinking water with vanadate was replaced daily with fresh vanadate solution. The diabetic group receiving no vanadate treatment was given daily SQ insulin injections (Lente I U/0.25 kg) during the last 10 days of pregnancy. Insulin treatment of diabetic pregnant dams at this level was not sufficient to normalize their blood glucose, which was evident from their persistent glycosuria. However, our previous experiments showed that these poorly controlled diabetic pregnant animals stood a much greater chance to carry pregnancy to term than untreated, overtly diabetic animals. Water intake was measured every day, and urine glucose from all diabetic animals was monitored daily for sugar using KetoDiastix. Periodic blood samples also were drawn to monitor blood glucose and vanadium levels (by graphite furnace atomic absorption spectroscopy) from days 10-20 of pregnancy. The dams were killed on day 20 of pregnancy, and blood samples were collected to determine serum vanadium and glucose levels. The fetuses from each pregnancy were cleaned, and the

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number of live pups and their total body weight were recorded for each pregnancy. Fetuses were killed by decapitation and the fetal blood from each pregnancy was pooled. The serum from the pooled fetal blood was saved for glucose and vanadium determinations. The uteri were examined for signs of abnormalities and/or embryo absorption. The ovaries and placentas were examined for gross abnormalities with a dissecting microscope..

Vanadium Assa~ Serum samples were diluted with 0.5% Triton X-100 and vanadium concentrations were determined by graphite furnace atomic absorption. Calibrations were performed by the Method of Standard Additions. Statistical Analysis For qualitative data, an overall chi-squared test of equality of proportions for independent samples was conducted to analyze these data to determine the statistical significance. For quantitative data, a linear regression model was used to determine the level of significance for the two vanadate dose levels, 0.00 and 0.25 mg/mL, with an indicator variable used to denote the diabetic and nondiabetic groups, and is shown below:

where: y is the quantitative variable being predicted, x I is the level of vanadate and x 2 is the indicator variable (0 nondiabetic, 1 diabetic). This allows potentially two separate lines to describe the effect of vanadate on the two groups (diabetic and nondiabetic), and allows statistical significance to be judged more easily. Additionally, two sample independent, sample t-tests (with nonpooled variances) were run to compare means of two specific groups. All statistical analyses were performed using MINITAB software for statistical analysis.

Results

The number of animals assigned to each of the four treatment groups was decided on the basis of the risk associated with each treatment. Eleven rats were assigned initially to the group with the highest risk, the diabetic animals on vanadate treatment. Oral vanadate treatment had a severely deleterious effect on the diabetic pregnant group. It is important to note that deaths occurred only in the diabetic (pregnant) group on vanadate treatment, and nowhere else. A total of only 6 animals from the original 1 I animals assigned to this group survived till day 20 of pregnancy. The probability of such a high level of attrition was estimated by using a chi square test for equality of proportions. The calculated chi-square value was 9.88, which was significant at the 0.05 confidence level (p-value of 0.02). This indicates, despite the smallness of the sample, that the diabetic pregnant group is more vulnerable to oral vanadate treatment. This conclusion is consistent with our observation in an earlier experiment to delineate the effect of oral vanadate treatment on reproductive efficiency. Although paired fed studies were not a part of the experimental design, the overall appearance and blood glucose values of the test animals on vanadate treatment did not suggest caloric depravation. The effects of oral vanadate treatment on different metabolic parameters and on blood vanadium levels for diabetic and nondiabetic pregnant dams during the last half of the pregnancy are summarized in Table 1. The blood vanadium levels in the different treatment groups of pregnant dams on day 20 of pregnancy is given in column 6 of this table. The regression analysis of the blood vanadium levels in the two vanadate-treated groups yielded an

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overall R2 value of 7 2 8 % , indicating a good fit of the data to the statistical model used. The analysis gave 2 lines with significantly positive slopes (with a p-value of 0.005), and slope values of 1177 and 2257 for nondiabetic and diabetic pregnant groups, respectively. These slope values were also significantly different at the 0.05 level (with a p-value of 0.054). The intercept values of 28.7 and 84.0 for the nondiabetic and the diabetic pregnant groups, respectively, were not statistically different from each other or from zero. In addition, the blood vanadium levels in diabetic pregnant rats on oral vanadate treatment were significantly higher (648 ± 233 vs 322 ± 190, MEAN ± SD) at the 0.05 level (with a p-value of 0.023), when compared to the same in normal pregnant dams on an identical vanadate regimen. These observations support the conclusion of significantly higher blood vanadium levels in the pregnant diabetic dams when compared to the same for pregnant nondiabetic dams on an identical vanadate regimen. Table 1. Effect o f Oral Vanadate Treatment on D a m s Group

Number of Animals

*Blood Glucose (mg/dL)

Unne Glucose mg/dL

UHne Ketone mg/dL

*Blood [V] ppb

*Fluid Intake mL/day

Nondiabetic Control

5

97 ± 25

Neg

Neg

28 ± 7

55 ± 16

Nondiabetic on Vanadate

7

82.5 ± 28.8

Neg

Neg

322 + 190

27 ±10

Diabetic Control

6

532 ± 115

2000

0 - 80

84 ± 68

191 ± 30

Diabetic on Vanadate

6

278 ± 114

2000 (5) Neg (1)

0 - 15

648 ± 233

63 ± 2 6

* Values are mean ± SD The data for daily water consumption by pregnant dams m different groups are presented in column 7 of Table 1. The effect of oral vanadate treatment on water consumption was analyzed using regression analysis. The analysis yielded 2 separate lines with negative slopes and slope values o f - 1 1 1 8 and -5127 for nondiabetic and diabetic pregnant dams, respectively. Both slopes also were significantly different from zero, and significantly different from each other with p-values of 0.041 and 0.001 for nondiabetic and diabetic groups, respectively. The overall R~ value of 91.1% indicates an extremely good fit of the data to the statistical model used. The data on water intake for the animals in different treatment groups suggest that water intake is significantly higher for the diabetic group at all points, irrespective of vanadate treatment, when compared to the same for the nondiabetic pregnant group. Furthermore, the data demonstrate a significant decrease in water intake in response to vanadate treatment, regardless of the metabolic status (diabetic or normal). Finally, water consumption for the diabetic group on oral vanadate treatment was approximately twice that of water consumption by the nondiabetic group on an identical vanadate regimen (63 -t: 26 vs 27 i 10, MEAN ± SD). These data indicate that for the vanadate-treated groups, the daily vanadate intake by the diabetic group was approximately twice that of the daily vanadate intake for the nondiabetic group. However, the presence of a significantly higher level of blood vanadium in the diabetic pregnant group compared to the blood vanadium level for the nondiabetic group on identical

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vanadate treatment may suggest an altered vanadium clearance by the diabetic pregnant group. Because of the dissimilar water intakes (vanadate intakes) and the lack of definitive pharmacokinetic data, a firm conclusion can not be made on this issue. It is of interest to point out that in a separate study in our laboratory, using male rats, oral vanadate treatment increased blood vanadium to similar levels in both diabetic (355 + 49, MEAN 4- SEM; N=6) and nondiabetic (335 ± 57, MEAN ± SEM; N=6) groups, despite a significantly higher rate of water (vanadate) intake by diabetic animals. The effect of oral vanadate treatment on the plasma glucose levels in different treatment groups also is presented in Table I. The data were analyzed using a two sample independent, sample t-test (with nonpooled variances). Oral vanadate treatment significantly lowered the blood glucose level in the diabetic pregnant group, when compared to that in the diabetic group without vanadate treatment (278 4- 114 vs 532 4- 115, MEAN ± SD; p =0.006). However, the blood glucose level in the vanadate-treated diabetic pregnant group still remained significantly higher than the blood glucose level in the nondiabetic pregnant control group (278 ± 114 vs 97 ± 2 5 , M E A N ± S D : p =0.013). The urine glucose level also remained high in the vanadate-treated diabetic group, indicating ineffectiveness of oral vanadate treatment in ameliorating hyperglycenna (Table 1). This observation is paradoxical, since an identical oral vanadate regimen was effective in normalizing blood glucose in diabetic male rats with a blood vanadium level of 355 4- 49, MEAN 4- SEM: N=6 which is significantly lower than the blood vanadium level in the vanadate-treated diabetic pregnant animals (648 ± 233, MEAN ± SD) at 0.05 level (p = 0.03). The data indicate that the ability of oral vanadate treatment to normalize blood glucose in diabetic pregnant animals is compromised significantly. Finally, all dams were weighed 2 times: at the time of assignment to treatment groups and at the time of initiation of treatment, day 10 of pregnancy. No si[mificant difference m weights were observed either between or withm groups.

Table 2. Effect of Vanadate Treatment on Pups

T~eatment Group

Number of Animals

Average Number of Pups

Average Mass of Pups

~Pup Blood [V] ppb

*Ratio [V] pup/dam

Nondiabetic Control

5

9.6

4.02

34 ± 26

1.2 ± 1.0

Nondiabetic on Vanadate

7

6.71

3.60

60 ± 23

0.19 + 0.13

Diabetic Control

6

11.3

3.11

56 ± 13

0.67 + 0.56

Diabetic on Vanadate

6

5.5

3.78

161 ± 23

0.25 ± 0.10

* Values are mean ± SD

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The data on the surviving pups (day 20 fetuses) are summarized in Table 2. Statistical analysis and determination of statistical significance were difficult to obtain for these data, since pooled samples of the entire litter per pregnancy were used in the various measurements. This, in addition to the low survival rate, effectively resulted in small samples to analyze. Although it was not possible to determine if the toxic effects on fetuses were direct or indirect, resultmg from toxicity to the dams, the conclusions that were significant and meaningful are listed below. One of the most consistent observations was the effect of maternal blood vanadium levels on the average number of fetuses on day 20 of pregnancy. Regression analysis showed that initiation of vanadate treatment of the dams on day 10 of pregnancy markedly reduced the number of fetuses per pregnancy in both nondiabetic and diabetic dams, when compared to the treatment groups that did not receive vanadate (p-value of 0.053) as indicated in Table 2. Because of the limitations mentioned above, no significant difference between the diabetic and the nondiabetic groups could be detected. 2.

The blood vanadium level in fetuses from vanadate-treated, diabetic pregnant dams was fotmd to be significantly higher at the 0.05 level of significance with a p-value of 0.0039 (161 ± 13 vs 60 ± 10, MEAN ± SEM), when compared to that of nondiabetic pregnant dams subjected to an identical vanadate regimen (Table 2). Again, because of the limitations mentioned earlier, the vanadium level in the pups' blood from the nondiabetic group treated with vanadate could not be differentiated significantly from that of either control group. However, given the decrease in the average number of pups from the nondiabetic, vanadate-treated group, this suggests that even a modest increase in vanadium levels can have a significant impact on the growth and development of the con ceptus.

3

The average pup mass was considered, but once again due to the aforementioned limitations, no significant differences could be detected

4.

Maternal blood vanadium levels had a significant correlation with the fetal blood vanadium levels in both nondiabetic and diabetic pregnant dams on a vanadate regimen. Fetal blood vanadium levels were found to be consistently about 20% of the matemal blood vanadium levels in vanadate-treated groups.

These observations suggest that a significant fraction ( - 2 0 % ) of vanadium in maternal blood enters fetal circulation and that vanadium, when present in fetal circulation even as late as the third trimester of pregnancy, has a negative effect on fetal growth and development.

Discussion Vanadate and vanadyl ions, as well as peroxovanadates, have been found to be effective in the management of diabetes-associated hyperglycemia in both Type I and Type II diabetes in different animal models (2,7,12,38). The use of oxovanadiums as a therapeutic agent in the management of diabetic hyperglycemia in humans is under serious consideration (2,8,38). Pregnancy is associated with increased insulin resistance principally due to increased levels of placental hormones, such as steroids and placental lactogens (39,40). The specific molecular mechanism(s) involved in reduced tissue sensitivity to insulin during pregnancy is not known. Since oral vanadate treatment was effective in normalizing blood sugar in the insulin-resistant, Type II diabetic animal models (6,8), we examined the effectiveness of oral vanadate treatment

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in the amelioration of hyperglycemia in diabetic pregnant dams during the second half of pregnancy because diabetic manifestations are more profound during this period (38,40,41) Rapidly growing cells and tissues are extremely vulnerable to the deleterious effects of various toxic metals and toxic compounds (32,33,42). Therefore, the impact of elevated levels of matemal blood vanadium on the conceptus during its rapid growing phase, the second half of the pregnancy, also was examined in both normal and diabetic pregnancy. Oral vanadate treatment had an acute and severely deleterious effect on the diabetic pregnant dams. Five of the 1 ! animals in this treatment group died within 6 days following initiation of oral vanadate treatment. The specific cause of death from oral vanadate treatment is not known at this time. The one most consistent observation in this study was the presence of a very high blood vanadium level in the diabetic pregnant dams on an oral vanadate regimen. The blood vanadium level in the diabetic pregnant dams was consistently twice that present in nondiabetic pregnant dams on an identical vanadate regimen. Oxovanadimns are biologically active and are toxic when present in high concentrations (1,2,36). Pregnancy, in itself, poses significant risks to the diabetic mother and to the conceptus (39-41,43) . Placental hormones, such as steroids and placental lactogens, are responsible for magnifying the risk in diabetic pregnancy by interjecting an additional risk factor, insulin resistance, that increases the severity of diabetes during pregnancy (39-41). High blood glucose and ketobodies have toxic effects in various tissues because they can increase free radical production (42,44). Oxovanadiums also can accelerate the production of various reactive oxygen species, as well as other highly reactive free radicals, such as O H (23-27). Thus, it is possible that increased production of free radicals in response to the combined effects of elevated levels of blood glucose, ketone bodies and blood vanadium in the diabetic pregnant group on vanadate treatment may be responsible for the observed high rate of attrition in this group. Data also show that oral vanadate treatment is not effective in normalizing blood sugar in diabetic pregnant dams (Table 1). Oral vanadate treatment was only partially effective in reducing blood glucose in the diabetic pregnanl animals, which suggests that at least some component(s) of pregnancy-associated insulin resistance is located at a site that is distal to those sites of vanadate action responsible for the normalization of diabetic hyperglycemia. Oral vanadate treatment controls hyperglycemia in diabetic animals by improving glucose uptake and utilization by the peripheral tissues, such as muscle and fat (8,38), and also by attenuating the increased levels of hepatic glucose production by the liver (9). Recently, it has been shown that pentavanadate, a product of vanadyl (VO 2+) and H202, can stimulate insulin receptor autophosphorylation and can stimulate glucose uptake in adipocytes (38). However, a direct application of vanadate was ineffective in producing similar effects in an identical system (38). Oral vanadate treatment, on the other hand, was quite effective in the induction of key insulin-dependent metabolic enzymes in diabetic rat livers (9). This suggests that different oxovanadiums are selective in their ability to interact with different metabolic pathways involved in insulin-mediated regulation of metabolism. Thus, it is possible that insulinantagonistic placental hormones can block only some of the effects of oxovanadiums when administered orally to diabetic pregnant animals. Oral vanadate treatment of both diabetic and nondiabetic pregnant dams had a neganve impact on the number of fetuses per pregnancy (Table 2). This could be a consequence of transplacental transfer of a significant amotmt of vanadate from the matemal to fetal compartments. As delineated in Table 2, the fetal blood vanadium level is about 20% of the maternal level in the vanadate-treated groups. Even though the blood vanadium level in the

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fetal compartment is lower than that in maternal blood, it still produced a significant effect on fetal development perhaps because of the fact that, as mentioned earlier, rapidly growing cells and tissues are extremely vulnerable to the deleterious effects of various toxic metals and toxic compounds. This negative effect of blood vanadium on fetal growth may be due to the generation of excess free radical and reactive species, since it has been documented that high levels of these species can result in embryo death (42,44). A careful examination of the placentas from different treatment groups under a dissecting microscope did not reveal any obvious abnormalities. However, a toxic effect of elevated vanadium levels in maternal blood on the placenta can not be ruled out. In this study, we have shown that oral vanadate treatment was ineffective in normalizing blood glucose in diabetic pregnancy. Additionally, administration of vanadate treatment caused an abnormally high level of vanadium in maternal and fetal blood, which resulted in several harmful effects on both the dam and conceptus. Therefore, consideration of the therapeutic use of oral vanadate treatment is contraindicated in the management of diabetes in females of reproductive age.

Acknowledgements The authors wish to thank the Indiana State University Research Committee and the Fratemal Order of Eagles Diabetes Fund for their support, in part, of this project.

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