TERATOLOGY 41:97-104 (1990)
Influence of Maternal Dietary Zinc Intake on In Vitro Tubulin Polymerization in Fetal Rat Brain PATRICIA I. OTEIZA, SUSANA CUELLAR, BO LONNERDAL, LUCILLE S. HURLEY, AND CARL L. KEEN Departments of Nutrition and Internal Medicine, University of California, Dauis, California 95616
ABSTRACT The hypothesis that one of the biochemical lesions underlying zinc deficiency-induced teratogenicity is altered microtubule formation was tested. Day 19 fetuses from zinc-deficient Sprague-Dawley dams were characterized by low brain supernate zinc concentrations and slow brain tubulin polymerization rates compared to controls. Brain supernate tubulin and protein concentrations were similar in zinc-deficient and control fetuses. In vitro brain tubulin polymerization rates were increased following addition of zinc to either control or zinc-deficient brain supernates; however, the stimulatory effect of added zinc on polymerization was significantly higher in brain supernates obtained from zinc-deficient fetuses compared to controls. These results support the idea that one effect of fetal zinc deficiency is a reduction in tubulin polymerization, which in turn may result in altered microtubule function. Severe zinc deficiency during development has been shown to be teratogenic in all experimental animals studied t o date (Apgar, '85; Keen and Hurley, '89). Typical malformations associated with severe zinc deficiency in the rat include cleft lip and palate, brain and eye malformations, and numerous abnormalities of the heart, lung, and urogenital system. Brain malformations observed include neural tube defects, hydrocephaly, exencephaly, and anencephaly (Rogers et al., '85; Dreosti, '89). While the teratogenicity of severe maternal zinc deficiency is well established, the mechanisms underlying these defects are not well understood. Several investigators have suggested that one biochemical lesion underlying the fetal abnormalities observed in zinc deficiency is abnormal nucleic acid metabolism (Dreosti et al., '85; Keen and Hurley, '89). DNA synthesis is significantly depressed in zinc-deficient embryos and fetuses compared to controls, and this lower rate of synthesis has been linked to low activities of DNA polymerase and thymidine kinase (Eckhert and Hurley, '77; Dreosti et al., '80). It has been argued that if the reductions in nucleic acid synthesis are severe, this could result in alterations in the differential rates of cellular growth neces0 1990 WILEY-LISS, INC
sary for normal morphogenesis. Dreosti ('87) has suggested that an additional biochemical lesion underlying the development of the abnormalities is an increased rate of cellular membrane lipid peroxidation, which results in membrane damage with resultant cellular necrosis and cell death, leading to asynchrony in development. Recently, we have investigated the hypothesis that abnormal cellular tubulin polymerization may also be a biochemical lesion underlying some of the defects associated with zinc deficiency. Zinc, at concentrations that are of physiological relevance, stimulates in vitro tubulin assembly into microtubules of normal morphological characteristics (Hesketh, '84), and tubulin assembly has been reported to be impaired in the brains of zinc-deficient adult rats (Hesketh, '81; Oteiza et al., '88). Given the multiple roles of tubulin in cellular metabolism, it is reasonable to suggest that zinc
Received March 8, 1989; accepted May 15, 1989. Address reprint requests to Dr. Carl L. Keen, Department of Nutrition, University of California, Davis, CA 95616. This paper is dedicated to Lucille S . Hurley. Her devotion to furthering our understanding of the role of nutrition in prenatal development has been an inspiration to us all.
98
P.I. OTEIZA E T AL.
deficiency-induced impairment of tubulin polymerization in embryonic and/or fetal tissue could result in marked developmental abnormalities. The current study was undertaken to determine the effects of maternal zinc deficiency on fetal brain tubulin polymerization and to determine if any observed alterations in tubulin polymerization could be corrected for by the addition of zinc to fetal brain supernates. MATERIALS AND METHODS
Animals and diets Adult female Sprague-Dawley rats weighing 200-225 g were purchased from a commercial source (Charles River Laboratories, Wilmington, MA). They were individually housed in stainless steel cages in a temperature (22-23'C) and photoperiod (12 h LID) controlled room. Animals were provided with distilled-deionized water ad libitum. All animals were allowed to acclimate to their environment for at least 7 d, during which time they were fed a complete purified diet containing 50 p.g Znig (Keen and Hurley, '89). Females were caged overnight with males of the same strain fed a nonpurified diet (Purina Rat Chow, Ralston Purina, Stockton, CA). Sperm plugs the following morning indicated a successful mating, and dams were considered to be a t d 0 of gestation a t this time. On d 0, rats were divided into three groups and fed one of the following diets throughout gestation: a) control diet (50 pg Znig diet) ad libitum (50 Zn AL); b) zinc-deficient diet (0.6 p.g Znig diet) ad libitum (0.6 Zn AL); or c) control diet given a t a restricted intake equal to that consumed by the zinc-deficient group (50 Zn RF). Initial maternal body weights were similar among the groups. Zinc concentration of the diets was verified by flame atomic absorption spectrophotometry (AAS; Instrumentation Laboratories, model 551). Food intake was recorded daily, and body weights were measured a t 5-d intervals. On d 19 of gestation, the dams were killed by overexposure to CO, and laparotomies were performed. Maternal blood was collected by cardiac puncture into syringes containing zinc-free heparin (Sarstedt, Princeton, NJ). Blood was centrifuged for 15 min a t 3,OOOg and the plasma was removed and stored frozen in acid-washed plastic vials until analyzed. Maternal livers and brains were removed and washed in cold saline solution
(0.9% NaC1) and frozen in plastic vials until analyzed. The gravid uterus was removed, the gravid uterine weight was recorded, and individual fetuses and placentas were then removed, freed of adhering membranes, examined, and weighed. All fetuses were examined for gross structural malformations. Fetal brain was removed and rinsed in ice-cold 0.1 M Pipes buffer (piperazine ethane sulfonic acid), pH 7.0, rolled on filter paper, and weighed. Brains from each litter were pooled and homogenized in Pipes buffer (25% whol). Homogenates were centrifuged at 100,OOOg for 30 min a t 4°C. Brain supernates were carefully decanted and used for the following determinations: tubulin polymerization, total protein concentration, trace element concentrations (zinc, copper, and iron), and tubulin concentration. Brain pellets were stored frozen for subsequent determination of trace elements (zinc, copper, and iron). Livers from each litter were also pooled, homogenized in Pipes buffer (20% wlvol), and centrifuged a t 100,OOOg for 30 min. Liver supernatant fluid and pellet were analyzed for zinc, copper, and iron concentrations.
Trace element determinations Zinc, copper, and iron concentrations in plasma, brain, and liver were determined by flame AAS as described by Clegg et al. ('81). Tubulin assembly Tubulin assembly was measured turbidimetrically (Timasheff and Grishman, '80; Oteiza et al., '88). The assembly was started by placing the samples in a UV-160 Shimadzu spectrophotometer with the sample holders a t 37°C. Absorbance at 350 nm was measured every 1-3 min over a 60-min period. The slope of the initial lineal part of the curve was calculated and considered to be the initial velocity of the reaction. When the effect of added zinc (final concentration 40 pM) or colchicine (final concentration 0.5 mM) on tubulin assembly was characterized, small aliquots of concentrated aqueous solutions of both solutes were added to the supernates immediately before incubating the samples at 37°C; the additions caused a dilution of 1part in 50 or less of the incubation mixture. In the presence of 0.5 mM colchicine (60 min incubation, 37"C), the supernate tubulin polymerization was 90% inhibited, establishing the validity of the assay for tubulin poly-
ZINC AND TUBULIN POLYMERIZATION
merization (Weisenberg et al., '68). Because of assay limitations, measurement of the effect of added zinc on tubulin assembly was only conducted on a limited number of samples.
30
" I
99
I
Y
Q)
20-
Y Determination of tubulin in 0 c brain supernates C Tubulin concentration in brain super- U nates was determined by electrophoresis in 8 1 0 polyacrylamide gels containing sodium L dodecyl sulfate using the discontinuous system described by Laemmli ('70). After the run, gels were fixed in a 10% trichloroacetic acid, 50% ethanol solution for 20 min, "0 5 10 15 20 stained with a solution containing 10% trichloroacetic acid, 30% ethanol, and 0.2% Day of Pregnancy Coomassie Brilliant Blue R-250 for 40 min, Fig. 1. Maternal food intake during gestation. Dams and destained with a 10% acetic acid, 40% were fed from day 0 to day 19 of gestation diets methanol solution. Gels were then scanned containing 50 pg Znig, ad libitum (A);50 pg Z d g , at a using a laser densitometer (Ultroscan XL, restricted intake ( 0 ) ;or 0.6 pg Znig, ad libitum (0).Food Pharmacia LKB). Tubulin concentration intake was recorded daily. was determined as a percentage of the total dye-binding capacity. B(ap)-tubulin from sea urchin sperm microtubules was used as diet supply in that spillage was minimal from day 3 through day 19. By day 4 of standard. pregnancy, food intake of the 0.6 Zn AL Protein concentration in brain supernates group was lower (P < .01) than that of the and total homogenates 50 Zn AL group (Fig. 1). On day 19 of Total protein concentration in brain ho- pregnancy (termination date), the rats in mogenates and supernates was determined the 50 Zn AL group consumed a n average of according to Lowry et al. ('51) using bovine 20.4 g dietlday while the average food intake in the 0.6 Zn AL group was 4.1 g serum albumin as a standard. dietlday (P < .01). Statistical analysis Similar to food intake, maternal weight One-way analysis of variance was used to gain was significantly affected by diet. The evaluate statistical significance (Statview 50 Zn AL group demonstrated a pronounced 512 + 1. The litter was used a s the statistical weight increase throughout pregnancy, unit for calculation of fetal values; thus, while the 0.6 Zn AL group tended to lose these values represent either means of litter weight over the course of the experiment. means within each group or means of litter Despite similar food intakes, weight gain in percentages as indicated. A probability of the 50 Zn RF group was slightly higher than .05 or less was considered significant. Post in the 0.6 Zn AL group. By day 19 of hoc contrasts were made using Fisher's test. pregnancy, mean weight changes were 107 2 4,25 2, and 3 2 3 g for the 50 Zn AL, 50 Chemicals Zn RF, and 0.6 Zn AL groups, respectively All chemicals were of the best grade (P < .005). available and were purchased from Sigma Reprod uctiue parameters and teratogenicity Chemical Co. (St. Louis, MO). Table 1 shows the results for the reproRESULTS ductive parameters examined in this study. Maternal food intake and weight gain Litter weight was significantly different Food intake of the 50 Zn RF group was among the groups, being highest in the 50 lower than in the 0.6 Zn AL groups on days Zn AL group and lowest in the 0.6 Zn AL 1and 2 of pregnancy because of food spillage group (P < .05). The number of live fetuses/ in these groups. By day 3 the animals in the litter as well as fetal weights were signifi50 Zn RF group adapted to the restricted cantly lower in the 0.6 Zn AL group comI
*
100
P.I. OTEIZA ET AL.
TABLE I . Reproductive parameters in dams fed diets with different Concentrations of zinc*,**,***
Live fetusesilitter Fetal weight (g) Placental weight ( g ) Placentaibody weight (%) Fetal brain weight (g) Brainibody weight (9%) Litters with malformations’ Malformed fetuses per litter (9%) Resorptions per litter (9%)’ Sites affected per litter (%’a)
13.1 5 0.4* 2.2 t 0.2* 0.38 2 0.01* 19 2 1 0.083 f 0.003* 4.0 5 0.2 Oil1 O* O* O*
12.4 i l.Ox:~** 1.7 -t O.l** 0.35 t 0.01* 21 t 2 0.077 ? 0.004* 4.6 f 0.2 2113 2.3 5 1.2* 7.6 4.6*,** 9.8 ? 4.6*
*
10.5 i 0.9”“ 1.5 t 0.1** 0.29 5 0.01** 21 2 1 0.064 ? 0.004** 4.4 2 0.3 16/17 61.1 i 8.3** 21.0 t 6.2** 64.6 2 7.4**
etected by external observation. g e t e c t e d by persistence of metrial nodes. Based on total sites. *,**,***Values are shown as mean k S.E.M. Data with different superscripts are statistically different (P< ,051by one-way ANOVA.
pared to the 50 Zn AL group (P < .05).Fetal weight was also adversely affected in the 50 Zn RF compared to the 50 Zn AL group. Placental and fetal brain weight were significantly lower (P < .05) in the 0.6 Zn AL group than in both 50 Zn groups on a n absolute basis; however, placental and brain weights were similar among the groups on a body weight basis. The number of litters that were adversely affected (based on the observation of at least one fetus with a gross abnormality) and the percentage of abnormal fetuses was different among groups; both parameters were markedly increased in the 0.6 Zn AL group, compared to the 50 Zn AL and 50 Zn RF groups. Resorption frequency and the percentage of sites affected were significantly higher (P < .05) in the 0.6 Zn AL group compared to the 50 Zn RF and 50 Zn AL groups. With regard to external malformations, two of the 13 litters were affected in the 50 Zn RF group; two of the 172 fetuses examined (1%)in this group had abnormalities (one incident of short tail and one incident of syndactyly). In the 0.6 Zn AL litters (17 litters examined), common malformations included cleft palate (70.6%), short tail (64.7%),microphthalmia (29.4%),exencephaly (23.5%), spina bifida cystica (17.6%), anencephaly (11.8%), missing digits (11.8%), no tail (11.8%), hydrocephaly (5.9%), and short jaws (5.9%). With regard to percentage of fetuses affected in the 0.6 Zn AL group (178 fetuses examined), the most common malformations were syndactyly (21.2%),short tail (18.4%),cleft palate (12.3%), and exencephaly (11.7%).
Trace element concentrations in maternal and fetal tissues Maternal plasma zinc concentration was significantly lower (P < .001) in the 0.6 Zn AL group than in the 50 Zn AL and 50 Zn RF groups (Table 2). Maternal plasma copper and iron concentrations were similar among groups. There were no significant differences in maternal brain zinc, copper, or iron concentrations among groups. Maternal liver copper and zinc concentrations were similar among groups (Table 2); however, liver iron concentrations in the 0.6 Zn AL dams were significantly higher than in the 50 Zn AL (P < .001) and 50 Zn RF dams (P < .005). Zinc concentration of the fetal brain 100,OOOg supernatant fluids was significantly different among the groups (Table 3). Zinc concentration in brain supernates of fetuses from dams fed the 50 Zn AL diet was significantly higher than fetal brain zinc supernate concentrations of the 0.6 Zn AL (P < .001) and 50 Zn RF groups (P < .05). Fetal brain supernate zinc concentration in the 50 Zn RF group was significantly higher than that of the 0.6 Zn AL group (P < .05). It should be noted that, given the amount of homogenizing buffer used for the brains, the values shown in Table 3 for “supernatant” zinc concentrations represent approximately a n 11-fold dilution of the “actual” brain supernatant fluid (i.e., the supernatant fluid that would be obtained if no homogenizing buffer was used). Thus, “actual” mean brain supernatant zinc concentrations for the 50 Zn AL, 50 Zn RF, and 0.6 Zn AL groups would be 0.153, 0.124, and
-t
4.4 t 0.4**
Fe 2.5 t 0.4" 2.7 t 0.3*
S.E.M. and are the average of 11 animals for the 50 Zn AL and 50 Zn
of dietary zinc during pregnancy' Liver Zn cu 0.37 t- 0.01 0.065 0.002 0.38 -t 0.01 0.072 t- 0.003 0.37 t 0.01 0.069 2 0.003
50 Zn AL 50 Zn RF 0.6 Zn AL
Zn 0.083 t- 0.004 0.081 f 0.005 0.080 -t 0.004
0.125 f0.007**
Zn
13.90 f 0.05* 11.30 -t 0.90** 9.00 t 0.90***
Fe 0.109 t 0.004* 0.097 i 0.004*
Fe 22.1 -t 1.9 18.8 t 2.3 21.1 -t 0.6
'The supernate zinc and iron concentrations shown have not been corrected for buffer dilution since the tubulin polymerization studies were conducted using "diluted brain supernates." Results are shown as mean -t S.E.M. for an N of 11, 11, and 17 for the 50 Zn AL, 50 Zn RF, and 0.6 Zn AL groups, respectively. *'**'***Data in the same column with different superscripts are significantly different, P
Influence of Maternal Dietary Zinc Intake on In Vitro Tubulin Polymerization in Fetal Rat Brain PATRICIA I. OTEIZA, SUSANA CUELLAR, BO LONNERDAL, LUCILLE S. HURLEY, AND CARL L. KEEN Departments of Nutrition and Internal Medicine, University of California, Dauis, California 95616
ABSTRACT The hypothesis that one of the biochemical lesions underlying zinc deficiency-induced teratogenicity is altered microtubule formation was tested. Day 19 fetuses from zinc-deficient Sprague-Dawley dams were characterized by low brain supernate zinc concentrations and slow brain tubulin polymerization rates compared to controls. Brain supernate tubulin and protein concentrations were similar in zinc-deficient and control fetuses. In vitro brain tubulin polymerization rates were increased following addition of zinc to either control or zinc-deficient brain supernates; however, the stimulatory effect of added zinc on polymerization was significantly higher in brain supernates obtained from zinc-deficient fetuses compared to controls. These results support the idea that one effect of fetal zinc deficiency is a reduction in tubulin polymerization, which in turn may result in altered microtubule function. Severe zinc deficiency during development has been shown to be teratogenic in all experimental animals studied t o date (Apgar, '85; Keen and Hurley, '89). Typical malformations associated with severe zinc deficiency in the rat include cleft lip and palate, brain and eye malformations, and numerous abnormalities of the heart, lung, and urogenital system. Brain malformations observed include neural tube defects, hydrocephaly, exencephaly, and anencephaly (Rogers et al., '85; Dreosti, '89). While the teratogenicity of severe maternal zinc deficiency is well established, the mechanisms underlying these defects are not well understood. Several investigators have suggested that one biochemical lesion underlying the fetal abnormalities observed in zinc deficiency is abnormal nucleic acid metabolism (Dreosti et al., '85; Keen and Hurley, '89). DNA synthesis is significantly depressed in zinc-deficient embryos and fetuses compared to controls, and this lower rate of synthesis has been linked to low activities of DNA polymerase and thymidine kinase (Eckhert and Hurley, '77; Dreosti et al., '80). It has been argued that if the reductions in nucleic acid synthesis are severe, this could result in alterations in the differential rates of cellular growth neces0 1990 WILEY-LISS, INC
sary for normal morphogenesis. Dreosti ('87) has suggested that an additional biochemical lesion underlying the development of the abnormalities is an increased rate of cellular membrane lipid peroxidation, which results in membrane damage with resultant cellular necrosis and cell death, leading to asynchrony in development. Recently, we have investigated the hypothesis that abnormal cellular tubulin polymerization may also be a biochemical lesion underlying some of the defects associated with zinc deficiency. Zinc, at concentrations that are of physiological relevance, stimulates in vitro tubulin assembly into microtubules of normal morphological characteristics (Hesketh, '84), and tubulin assembly has been reported to be impaired in the brains of zinc-deficient adult rats (Hesketh, '81; Oteiza et al., '88). Given the multiple roles of tubulin in cellular metabolism, it is reasonable to suggest that zinc
Received March 8, 1989; accepted May 15, 1989. Address reprint requests to Dr. Carl L. Keen, Department of Nutrition, University of California, Davis, CA 95616. This paper is dedicated to Lucille S . Hurley. Her devotion to furthering our understanding of the role of nutrition in prenatal development has been an inspiration to us all.
98
P.I. OTEIZA E T AL.
deficiency-induced impairment of tubulin polymerization in embryonic and/or fetal tissue could result in marked developmental abnormalities. The current study was undertaken to determine the effects of maternal zinc deficiency on fetal brain tubulin polymerization and to determine if any observed alterations in tubulin polymerization could be corrected for by the addition of zinc to fetal brain supernates. MATERIALS AND METHODS
Animals and diets Adult female Sprague-Dawley rats weighing 200-225 g were purchased from a commercial source (Charles River Laboratories, Wilmington, MA). They were individually housed in stainless steel cages in a temperature (22-23'C) and photoperiod (12 h LID) controlled room. Animals were provided with distilled-deionized water ad libitum. All animals were allowed to acclimate to their environment for at least 7 d, during which time they were fed a complete purified diet containing 50 p.g Znig (Keen and Hurley, '89). Females were caged overnight with males of the same strain fed a nonpurified diet (Purina Rat Chow, Ralston Purina, Stockton, CA). Sperm plugs the following morning indicated a successful mating, and dams were considered to be a t d 0 of gestation a t this time. On d 0, rats were divided into three groups and fed one of the following diets throughout gestation: a) control diet (50 pg Znig diet) ad libitum (50 Zn AL); b) zinc-deficient diet (0.6 p.g Znig diet) ad libitum (0.6 Zn AL); or c) control diet given a t a restricted intake equal to that consumed by the zinc-deficient group (50 Zn RF). Initial maternal body weights were similar among the groups. Zinc concentration of the diets was verified by flame atomic absorption spectrophotometry (AAS; Instrumentation Laboratories, model 551). Food intake was recorded daily, and body weights were measured a t 5-d intervals. On d 19 of gestation, the dams were killed by overexposure to CO, and laparotomies were performed. Maternal blood was collected by cardiac puncture into syringes containing zinc-free heparin (Sarstedt, Princeton, NJ). Blood was centrifuged for 15 min a t 3,OOOg and the plasma was removed and stored frozen in acid-washed plastic vials until analyzed. Maternal livers and brains were removed and washed in cold saline solution
(0.9% NaC1) and frozen in plastic vials until analyzed. The gravid uterus was removed, the gravid uterine weight was recorded, and individual fetuses and placentas were then removed, freed of adhering membranes, examined, and weighed. All fetuses were examined for gross structural malformations. Fetal brain was removed and rinsed in ice-cold 0.1 M Pipes buffer (piperazine ethane sulfonic acid), pH 7.0, rolled on filter paper, and weighed. Brains from each litter were pooled and homogenized in Pipes buffer (25% whol). Homogenates were centrifuged at 100,OOOg for 30 min a t 4°C. Brain supernates were carefully decanted and used for the following determinations: tubulin polymerization, total protein concentration, trace element concentrations (zinc, copper, and iron), and tubulin concentration. Brain pellets were stored frozen for subsequent determination of trace elements (zinc, copper, and iron). Livers from each litter were also pooled, homogenized in Pipes buffer (20% wlvol), and centrifuged a t 100,OOOg for 30 min. Liver supernatant fluid and pellet were analyzed for zinc, copper, and iron concentrations.
Trace element determinations Zinc, copper, and iron concentrations in plasma, brain, and liver were determined by flame AAS as described by Clegg et al. ('81). Tubulin assembly Tubulin assembly was measured turbidimetrically (Timasheff and Grishman, '80; Oteiza et al., '88). The assembly was started by placing the samples in a UV-160 Shimadzu spectrophotometer with the sample holders a t 37°C. Absorbance at 350 nm was measured every 1-3 min over a 60-min period. The slope of the initial lineal part of the curve was calculated and considered to be the initial velocity of the reaction. When the effect of added zinc (final concentration 40 pM) or colchicine (final concentration 0.5 mM) on tubulin assembly was characterized, small aliquots of concentrated aqueous solutions of both solutes were added to the supernates immediately before incubating the samples at 37°C; the additions caused a dilution of 1part in 50 or less of the incubation mixture. In the presence of 0.5 mM colchicine (60 min incubation, 37"C), the supernate tubulin polymerization was 90% inhibited, establishing the validity of the assay for tubulin poly-
ZINC AND TUBULIN POLYMERIZATION
merization (Weisenberg et al., '68). Because of assay limitations, measurement of the effect of added zinc on tubulin assembly was only conducted on a limited number of samples.
30
" I
99
I
Y
Q)
20-
Y Determination of tubulin in 0 c brain supernates C Tubulin concentration in brain super- U nates was determined by electrophoresis in 8 1 0 polyacrylamide gels containing sodium L dodecyl sulfate using the discontinuous system described by Laemmli ('70). After the run, gels were fixed in a 10% trichloroacetic acid, 50% ethanol solution for 20 min, "0 5 10 15 20 stained with a solution containing 10% trichloroacetic acid, 30% ethanol, and 0.2% Day of Pregnancy Coomassie Brilliant Blue R-250 for 40 min, Fig. 1. Maternal food intake during gestation. Dams and destained with a 10% acetic acid, 40% were fed from day 0 to day 19 of gestation diets methanol solution. Gels were then scanned containing 50 pg Znig, ad libitum (A);50 pg Z d g , at a using a laser densitometer (Ultroscan XL, restricted intake ( 0 ) ;or 0.6 pg Znig, ad libitum (0).Food Pharmacia LKB). Tubulin concentration intake was recorded daily. was determined as a percentage of the total dye-binding capacity. B(ap)-tubulin from sea urchin sperm microtubules was used as diet supply in that spillage was minimal from day 3 through day 19. By day 4 of standard. pregnancy, food intake of the 0.6 Zn AL Protein concentration in brain supernates group was lower (P < .01) than that of the and total homogenates 50 Zn AL group (Fig. 1). On day 19 of Total protein concentration in brain ho- pregnancy (termination date), the rats in mogenates and supernates was determined the 50 Zn AL group consumed a n average of according to Lowry et al. ('51) using bovine 20.4 g dietlday while the average food intake in the 0.6 Zn AL group was 4.1 g serum albumin as a standard. dietlday (P < .01). Statistical analysis Similar to food intake, maternal weight One-way analysis of variance was used to gain was significantly affected by diet. The evaluate statistical significance (Statview 50 Zn AL group demonstrated a pronounced 512 + 1. The litter was used a s the statistical weight increase throughout pregnancy, unit for calculation of fetal values; thus, while the 0.6 Zn AL group tended to lose these values represent either means of litter weight over the course of the experiment. means within each group or means of litter Despite similar food intakes, weight gain in percentages as indicated. A probability of the 50 Zn RF group was slightly higher than .05 or less was considered significant. Post in the 0.6 Zn AL group. By day 19 of hoc contrasts were made using Fisher's test. pregnancy, mean weight changes were 107 2 4,25 2, and 3 2 3 g for the 50 Zn AL, 50 Chemicals Zn RF, and 0.6 Zn AL groups, respectively All chemicals were of the best grade (P < .005). available and were purchased from Sigma Reprod uctiue parameters and teratogenicity Chemical Co. (St. Louis, MO). Table 1 shows the results for the reproRESULTS ductive parameters examined in this study. Maternal food intake and weight gain Litter weight was significantly different Food intake of the 50 Zn RF group was among the groups, being highest in the 50 lower than in the 0.6 Zn AL groups on days Zn AL group and lowest in the 0.6 Zn AL 1and 2 of pregnancy because of food spillage group (P < .05). The number of live fetuses/ in these groups. By day 3 the animals in the litter as well as fetal weights were signifi50 Zn RF group adapted to the restricted cantly lower in the 0.6 Zn AL group comI
*
100
P.I. OTEIZA ET AL.
TABLE I . Reproductive parameters in dams fed diets with different Concentrations of zinc*,**,***
Live fetusesilitter Fetal weight (g) Placental weight ( g ) Placentaibody weight (%) Fetal brain weight (g) Brainibody weight (9%) Litters with malformations’ Malformed fetuses per litter (9%) Resorptions per litter (9%)’ Sites affected per litter (%’a)
13.1 5 0.4* 2.2 t 0.2* 0.38 2 0.01* 19 2 1 0.083 f 0.003* 4.0 5 0.2 Oil1 O* O* O*
12.4 i l.Ox:~** 1.7 -t O.l** 0.35 t 0.01* 21 t 2 0.077 ? 0.004* 4.6 f 0.2 2113 2.3 5 1.2* 7.6 4.6*,** 9.8 ? 4.6*
*
10.5 i 0.9”“ 1.5 t 0.1** 0.29 5 0.01** 21 2 1 0.064 ? 0.004** 4.4 2 0.3 16/17 61.1 i 8.3** 21.0 t 6.2** 64.6 2 7.4**
etected by external observation. g e t e c t e d by persistence of metrial nodes. Based on total sites. *,**,***Values are shown as mean k S.E.M. Data with different superscripts are statistically different (P< ,051by one-way ANOVA.
pared to the 50 Zn AL group (P < .05).Fetal weight was also adversely affected in the 50 Zn RF compared to the 50 Zn AL group. Placental and fetal brain weight were significantly lower (P < .05) in the 0.6 Zn AL group than in both 50 Zn groups on a n absolute basis; however, placental and brain weights were similar among the groups on a body weight basis. The number of litters that were adversely affected (based on the observation of at least one fetus with a gross abnormality) and the percentage of abnormal fetuses was different among groups; both parameters were markedly increased in the 0.6 Zn AL group, compared to the 50 Zn AL and 50 Zn RF groups. Resorption frequency and the percentage of sites affected were significantly higher (P < .05) in the 0.6 Zn AL group compared to the 50 Zn RF and 50 Zn AL groups. With regard to external malformations, two of the 13 litters were affected in the 50 Zn RF group; two of the 172 fetuses examined (1%)in this group had abnormalities (one incident of short tail and one incident of syndactyly). In the 0.6 Zn AL litters (17 litters examined), common malformations included cleft palate (70.6%), short tail (64.7%),microphthalmia (29.4%),exencephaly (23.5%), spina bifida cystica (17.6%), anencephaly (11.8%), missing digits (11.8%), no tail (11.8%), hydrocephaly (5.9%), and short jaws (5.9%). With regard to percentage of fetuses affected in the 0.6 Zn AL group (178 fetuses examined), the most common malformations were syndactyly (21.2%),short tail (18.4%),cleft palate (12.3%), and exencephaly (11.7%).
Trace element concentrations in maternal and fetal tissues Maternal plasma zinc concentration was significantly lower (P < .001) in the 0.6 Zn AL group than in the 50 Zn AL and 50 Zn RF groups (Table 2). Maternal plasma copper and iron concentrations were similar among groups. There were no significant differences in maternal brain zinc, copper, or iron concentrations among groups. Maternal liver copper and zinc concentrations were similar among groups (Table 2); however, liver iron concentrations in the 0.6 Zn AL dams were significantly higher than in the 50 Zn AL (P < .001) and 50 Zn RF dams (P < .005). Zinc concentration of the fetal brain 100,OOOg supernatant fluids was significantly different among the groups (Table 3). Zinc concentration in brain supernates of fetuses from dams fed the 50 Zn AL diet was significantly higher than fetal brain zinc supernate concentrations of the 0.6 Zn AL (P < .001) and 50 Zn RF groups (P < .05). Fetal brain supernate zinc concentration in the 50 Zn RF group was significantly higher than that of the 0.6 Zn AL group (P < .05). It should be noted that, given the amount of homogenizing buffer used for the brains, the values shown in Table 3 for “supernatant” zinc concentrations represent approximately a n 11-fold dilution of the “actual” brain supernatant fluid (i.e., the supernatant fluid that would be obtained if no homogenizing buffer was used). Thus, “actual” mean brain supernatant zinc concentrations for the 50 Zn AL, 50 Zn RF, and 0.6 Zn AL groups would be 0.153, 0.124, and
-t
4.4 t 0.4**
Fe 2.5 t 0.4" 2.7 t 0.3*
S.E.M. and are the average of 11 animals for the 50 Zn AL and 50 Zn
of dietary zinc during pregnancy' Liver Zn cu 0.37 t- 0.01 0.065 0.002 0.38 -t 0.01 0.072 t- 0.003 0.37 t 0.01 0.069 2 0.003
50 Zn AL 50 Zn RF 0.6 Zn AL
Zn 0.083 t- 0.004 0.081 f 0.005 0.080 -t 0.004
0.125 f0.007**
Zn
13.90 f 0.05* 11.30 -t 0.90** 9.00 t 0.90***
Fe 0.109 t 0.004* 0.097 i 0.004*
Fe 22.1 -t 1.9 18.8 t 2.3 21.1 -t 0.6
'The supernate zinc and iron concentrations shown have not been corrected for buffer dilution since the tubulin polymerization studies were conducted using "diluted brain supernates." Results are shown as mean -t S.E.M. for an N of 11, 11, and 17 for the 50 Zn AL, 50 Zn RF, and 0.6 Zn AL groups, respectively. *'**'***Data in the same column with different superscripts are significantly different, P
