vitamins c and e improve rat embryonic antioxidant defense mechanism in diabetic culture medium

Vitamins C and E Improve Rat EmbryonicAntioxidant Defense Mechanism in DiabeticCulture MediumVARDA ZAKEN,1 RON KOHEN,2 AND ASHER ORNOY1*1Department of Anatomy and Cell Biology and 2Department of Pharmacy, The Hebrew UniversityHadassah Medical School, Jerusalem, Israel

ABSTRACT

Background: Diabetes teratogenicity seems to berelated to embryonic oxidative stress and the extent ofthe embryonic damage can apparently be reduced byantioxidants. We have studied the mechanism by whichantioxidants, such as vitamins C and E, reduce diabe-tes-induced embryonic damage. We therefore com-pared the antioxidant capacity of 10.5-day-old rat em-bryos and their yolk sacs cultured for 28h in diabeticculture medium with or without vitamins C and E.

Methods: The embryos were cultured in 90% rat se-rum to which 2mg/ml glucose, 2mg/ml beta hydroxybutyrate (BHOB) and 10mg/ml of acetoacetate wereadded. Rat embryos were also cultured in a diabeticmedium with 25mg/ml of vitamin E and 50mg/ml ofvitamin C. Control embryos were cultured in normal ratserum with or without vitamins C and E.

Results: Decreased activity of Cu/Zn superoxide dis-mutase (SOD) and of catalase (CAT) in the “diabetic”embryos and their yolk sacs, and reduced concentra-tions of low molecular weight antioxidant (LMWA) werefound. Under these conditions we also found a de-crease in vitamin C and vitamin E concentrations in theembryos, as measured by HPLC. In situ hybridizationfor SOD mRNA showed a marked reduction of SODmRNA in the brain, spinal cord, heart and liver ofembryos cultured in diabetic medium in comparison tocontrols. Following the addition of vitamins C and E tothe diabetic culture medium, SOD and CAT activity, theconcentrations of LMWA, the levels of vitamin C and Eand the expression of SOD mRNA in the embryos andyolk sacs returned to normal.

Conclusions: Diabetic metabolic factors seem to havea direct effect on embryonic SOD gene and perhapsgenes of other antioxidant enzymes, reducing embry-onic endogenous antioxidant defense mechanism. Thisin turn may cause a depletion of the LMWA, such asvitamins C and E. The addition of these vitamins nor-malizes the embryonic antioxidant defense mecha-nism, reducing the damage caused by the diabeticenvironment.

Teratology 64:33–44, 2001. © 2001 Wiley-Liss, Inc.

INTRODUCTION

Reactive oxygen species (ROS) are defined as sub-stances that possess one or more unpaired electrons.The body utilizes antioxidant reserves to cope withoxidative stress. Antioxidant defense mechanism in-cludes scavenging antioxidants that remove ROS onceformed, thus preventing free radical chain reactions.They are composed of: 1) Enzymes e.g. superoxide dis-mutase, (SOD) glutathione peroxidase (GSH-Px), cata-lase, (CAT) (Chevion, ’88; Hallivell, ’90; Hass et al., ’89;Trocino et al., ’95). 2) Lipophilic and hydrophilic low-molecular-weight antioxidants (LMWA) e.g. Glutha-tione, Ascorbate (vitamin C), a Tocopherol (vitamin E),bilirubin, uric acid, carotenoids (vitamin A) and fla-vonoids. (Sharma and Buetner, ’93). 3) Repair enzymesthat repair or remove ROS-damaged biomolecules.These include DNA repair enzymes and methioninesulfoxide reductase (Hallivel, ’90).

Of the lipid soluble antioxidants, vitamin E (a To-copherol) seems to play the most important role as itprevents lipid peroxidation by donating hydrogen tosuperoxide radicals (Sharma & Buetner, ’93). In thatreaction vitamin E is converted to a weak free radical(a Tocopherol radical) that may be converted back to atocopherol in redox cycle reactions involving vitamin Cand/or coenzyme Q (Sharma & Buetner, ’93; Wang &Quinn, ’99, ’00). The combination of vitamins C and Eas antioxidants is therefore very important in the pre-vention of lipid peroxidation.

ROS are thought to be involved in the etiology ofnumerous diseases, such as arteriosclerotic cardiovas-cular disease, ischemic injuries and aging processes(Gutteridge, ’93; Hallivell, ’90; Halliwell et al., ’92; Hal-liwell and Gutteridge, ’95). They are produced in largeamounts in diabetes and apparently in various meta-

R.K. is affiliated with the David R. Bloom Center of Pharmacy (since2000).

Supported by grant No 032-5196 from the Israel Science Foundation.This study served for partial fulfillment of the requirements for a PhD from the Hebrew University.

*Correspondence: Asher Ornoy, Department of Anatomy and CellBiology, The Hebrew University, Hadassah Medical School, Jerusa-lem, P.O. Box 1227, Jerusalem, 91120 Israel.E-mail: [email protected].

TERATOLOGY 64:33–44 (2001)

© 2001 WILEY-LISS, INC.

bolic disorders (Papaccio et al., ’86). Genetic and met-abolic factors are involved in the etiology of diabetesinduced teratogenicity. High levels of glucose, acetoac-etate and beta-hydroxybutyrate (B-HOB) in diabetesincreased the production of ROS (Eriksson, ’91; Erikssonand Borg, ’93; Ornoy et al., ’96). These ROS are believedto have an important role in the etiology of diabetesinduced congenital anomalies (Eriksson, ’91; Erikssonand Borg, ’93; Ornoy et al., ’96; Wentzel et al., ’97).

In previous studies of the presence of LMWA in 2- to4-cell-stage mouse embryos and in blastocysts both invivo and in vitro, we found a higher level of LMWA inthe blastocysts as compared with the 2- to 4-cell-stageembryo (Ornoy et al., ’96). We then studied the activityof SOD and CAT in 9.5 to 12.5-day-old rat embryos invivo and in vitro. A gradual increase in the activity ofboth enzymes was found with the advancement of em-bryonic age. Cyclic voltammetry (CV) measurementsrevealed an increase in oxidation potential and in theconcentration of antioxidants in embryos and yolk sacswith the increase in embryonic age. No difference wasfound in the various parameters between embryos cul-tured in vivo and in vitro (Zaken et al., ’99). Otherinvestigators have shown that the activity of SOD,CAT, and GSH Px increases with embryonic age,reaching its peak during the neonatal period (Allen andBalin, ’89; Frank, ’91). The relative immaturity of theantioxidant defense mechanism predisposes the em-bryo to a heightened risk of oxidative attacks on pro-teins, DNA and lipid membranes (Fantel et al., ’92).

Eriksson (’91) found that the addition of SOD to“diabetic culture medium” reduced the rate of anoma-lies in 10.5-day-old rat embryos in culture. Erikssonand Borg (’93) found that the addition of antioxidantsto the culture medium also reduced the rate of embry-onic anomalies. Cederberg and Eriksson (’97) found areduction in CAT activity in 11-day-old diabetic ratembryos and Sivan et al. (’97) reported on reduced SODactivity in 11.5-day-old diabetic rat embryos with neu-ral tube defects.

In a previous study we cultured 10.5-day-old ratembryos for 28h in rat serum containing glucose (3mg/ml) BHOB (2mg/ml) and aceto-acetate (10mg/ml). Un-der these diabetic conditions, 71.3% of embryos hadanomalies. We found a significant decrease in the ac-tivity of SOD and CAT in the embryos and their yolksacs in comparison to controls as well as a markedreduction or absence of an entire group of LMWA. Inaddition we found a decrease in the concentration ofthe LMWA, vitamins C and E (Ornoy et al., ’99).

The activities of the antioxidant enzymes and theconcentrations of the LMWA were lower in the mal-formed “diabetic” embryos in comparison to “diabetic”embryos without anomalies. In this previous study(Ornoy et al., ’99), we also found that the addition ofvitamins C and E to the “diabetic” medium signifi-cantly reduced the damage to the embryos. It increasedthe percent of normal living embryos to 86.7%, as op-posed to only 28.6% normal embryos in those cultured

in diabetic serum and normalized embryonic and yolksac growth and protein content (Ornoy et al., ’99).

The purpose of the present study was to assess theantioxidant defense mechanism in these cultured em-bryos under the influence of antioxidants. We were alsointerested to assess whether the changes in the anti-oxidant defense mechanism induced by diabetic cultureconditions result from genomic effects of the diabeticenvironment. We also studied the SOD gene expression(SOD mRNA) using in situ hybridization.

MATERIALS AND METHODS

Six-eight weeks old female rats of the Hebrew Uni-versity Sabra strain were mated for 10–12 h overnight.The day sperms found in the vagina was consideredday 0. All normal-appearing 10 days old rat embryoswere removed for whole embryo culture according tothe method described by New (’78). The details of theexperimental methodology were published elsewhere(Ornoy et al., ’99). Each embryo was cultured in 0.9 mlrat serum and 0.1 ml distilled water, 4 embryos per oneculture bottle (Zusman and Ornoy, ’87). The “diabetic”embryos were cultured in 90% rat serum to which 2mg/ml glucose, 2 mg/ml BHOB (acid, Sigma) and 10mg/ml of acetoacetate (sodium salt, Sigma) were added,comprising the “diabetic environment.” These concen-trations of metabolites were chosen as our previousstudies demonstrated that these are the lowest levelsof combined “diabetic” metabolites that are teratogenicto rat embryos in culture (Zusman and Ornoy, ’87). Inaddition we cultured rat embryos in “diabetic” mediumto which we added 50 mg/ml of vitamin C, (Fluka) and25 mg/ml of vitamin E (water soluble Trolox, Aldrich).These concentrations were chosen as they are 1–3times the concentration in human blood (Lockitch etal., ’88). Embryos cultured in 90% control rat serum orin 90% rat serum with Vitamins C and E served as twocontrol groups. Embryos from the same dam were ran-domly divided for culture in control and diabetic me-dium. During the first day of culture we added thefollowing gas mixtures: 20%O2, 75%N2 5%CO2. On thesecond day the following gas mixture was used (for 4hours only): 40%O2, 55%N2, 5%CO2. The culture tem-perature was 37°C. We used for culture a Stuart Sci-entific Incubator, S.I.60 D. UK with a rotator. Follow-ing culture, only the embryos with beating hearts andintact yolk sac circulation (living embryos) were scoredaccording to the method described by Brown and Fabro(’81) and screened for individual anomalies as de-scribed by us previously (Zusman et al., ’87; Ornoy etal., ’99). Embryos and yolk sacs were then homogenized(each embryo and/or yolk sac in 1 ml PBS) using a VortexHomogenizer and stored in 270°C, until further study.

Protein determination: This was performed accordingto the method described by Bradford (’76) i.e. spectropho-tometric determination of the complex of protein and thereagent (dye with phosphoric acid and methanol).

Studies of the Antioxidant Defense Mechanism: Ineach embryo and/or yolk sac we examined the activity

34 ZAKEN ET AL.

of SOD and of CAT and the LMWA content using CVand HPLC. Results were expressed per mg protein ofthe embryos and yolk sacs.

Cyclic voltammetry

The total reducing power of the embryos and/or yolksacs was studied on water-soluble homogenates. Themethod was described in details by Kohen and by oth-ers (Chevion et al., ’97; Guadalupe et al., ’92; Herbac &Kohen, ’00; Kohen, ’92; Kohen, ’93; Kohen et al., ’97;Kohen et al., ’00), and by us previously (Ornoy et al.,’99). In brief, samples were prepared for CV analysis asfollows: embryos and yolk sacs were homogenized in 1ml of 0.1 M phosphate buffered-saline (PBS), pH 7.2.Following homogenization, the samples were centri-fuged at 1000 g for 10 minutes at 4°C to remove large,insoluble particles. Aliquots were removed for proteindetermination and the remaining samples were used tomeasure water-soluble LMWA as described previously(Ornoy et al., ’99).

CV can provide information regarding the kinetics ofthe electron transfer step and often information aboutthe mechanism of redox changes in the studied system.The tested solution is subjected to the potential im-posed on electrodes which is linearly swept from ini-tially to vertex potential (forward scan) and then sweptback to final potential (reverse scan) usually of themagnitude as the initial potential. One or more cyclescan be carried out if only half the cycle is used. Thetechnique is referred to as linear sweep voltammetry.The result of the CV experiment is the potential–cur-rent curve, the cyclic voltammogram (Herbac andKohen, ’00). The potential imposed on the electrode isaimed at oxidizing or reducing species present in thevoltammetric cell.

A BAS model CV-1B cyclic voltammeter (West Lafay-ette, Inc. USA) was used to evaluate the reducingpower of the tissue samples (Halliwell, ’90). The cyclicvoltammeters use 3 electrodes: the working electrodethat is a glassy carbon disk (BAS MF-2012) of 3.2 mmdiameter; a platinum wire serving as the counter elec-trode and the reference electrode. Cyclic voltammo-grams (CV) were recorded at a range of 0–1.3 V and ata rate of 100 mV/s, vs. an Ag/AgCl reference electrode.The working electrode was polished prior to each mea-surement.

CV tracings were analyzed to determine peak poten-tial and anodic current. The anodic current (Ia) corre-lated with the concentration of the reducing equiva-lents. The peak potential (the capacity of LMWA totransfer electrons) is calculated from the potential ap-plied on the working electrode as measured in volts onthe X-axis. This potential is calculated as one-half ofthe increase in the current of the anodic wave andrepresents the ability of the reducing equivalents inthe embryo or yolk sac to donate their electrons to theworking electrode. One or several LMWA possessing aspecific peak potential are recorded in one wave. Thelower the potential, the higher is the ability of theLMWA to donate their electrons, indicating stronger

reducing power (better antioxidant capacity). At leastthree tracings were recorded for each sample, and therepeated scans yielded similar CV tracing. The detec-tion limit of each peak potential is 50 mV; a change of .50 mV in peak potential (above the detection limit ofthe cyclic voltammeter) was considered significant. Itwas therefore attributed to the presence of anotherreducing equivalent or group of LMWA, possessing adifferent peak potential. The buffer did not possess anyreducing properties, as it did not show any anodic waveup to 1.3 V (results not shown). The concentration ofLMWA was measured as the anodic current in mA onthe Y-axis. It directly and linearly correlates with theconcentration of the LMWA. The detection limit on theY-axis it is 1.0mA, detecting a minimal concentration of1 mM.

A missing wave means that the specific group ofLMWA is very low, below the detection level, or en-tirely missing (Kohen, ’93; Kohen et al., ’00; Ornoy etal., ’99). Measurements were carried out at 37°C in 0.01M phosphate buffered saline, pH 7.4.

HPLC-ECD measurements

To evaluate the various compounds composing thecyclic voltammeter waves we first studied the water-soluble fraction. Homogenized tissue samples weretreated with 25% TCA to precipitate out proteins; 20mlof deproteinized samples was injected into an HPLCsystem (Kontron, Switzerland). The column was con-nected to a LC4A amperometric electrochemical detec-tor (BAS West Lafayette IN). A mobile phase consistingof 40 mM sodium acetate buffer, pH 4.75, 0.54 mMNa2EDTA and 1.5 mM tetrabutylammonium hydroxidewas used for analysis of vitamin C and uric acid. Forvitamin A analysis we extracted the lypophylic LMWAfrom the homogenates by dissolving the tissue samplesin methanol:hexane (1:4) and recentrifugation at 1,000gfor 10 min. The upper and lower layers were separated,the organic solvents were evaporated and the residuewas dissolved in acetonitrile:methanol (1:1) containing1% tetrabutylammonium perchlorate. We used a mo-bile phase containing 20 mM lithium perchlorate inmethanol:ethanol:isopropanol for the analysis of vita-min A (Motchnik et al., ’94). Standards (ubiquinol-10)were prepared according to Yamashita and Yamamoto(’97). Standard curves were prepared for each stan-dard, as described by us previously (Ornoy et al., ’99).

Detection of antioxidant enzyme activities

Cu/Zn activity. Cu/Zn SOD activity was studied bythe method described by McCord and Friedovich (’88)and by Grankvist et al. (’81). Xanthine oxidase (1 U/ml(Sigma) added to the solution containing hypoxanthine(6 mM) produces ROS. Cytochrome C, 3mM (Sigma)was used as a detector. The superoxide radicals reducethe cytochrome C, and the reduced cytochrome C ismeasured by spectrophotometry (Uvicon 933, Kontron,Switzerland) at 550nm. SOD present in the samplesreduces the amount of superoxide radicals, thereforereducing the amount of reduced cytochrome C detected

VITAMINS C AND E REDUCE DIABETIC EMBRYONIC DAMAGE 35

by the spectrophotometer. The incubation solution wasPBS. The assay was conducted at 25°C without anypre-incubation.

CAT activity. The method was described by Thur-man et al. (’72). Aliquots of 15 or 20 ml of the homoge-nate were added to the reaction mixture containinghydrogen peroxide at a concentration of 75 mM. Thereaction mixture also contained 3 Amino -1,2,4, triazole(Sigma) 1 mM to stop glutathione peroxidase activity.Following 10 minutes of incubation at room tempera-ture the reaction was stopped by addition of 200 mltrichloroacetic acid 30% (TCA) and the remaining hy-drogen peroxide was determined according to the Thur-man procedure (Thurman et al., ’72). In brief, it mea-sures the red complex that is formed by hydrogenperoxide, ferrous ammonium sulfate (Sigma) and 25%thiocyanate (Sigma). The concentration of the complex,which is directly related to the concentration of hydro-gen peroxide in the tested solution, is read by a spec-trophotometer at 480 nm. Details of SOD and CATdeterminations was described by us previously (Ornoyet al., ’99). Enzymatic activity (the time for the reac-tion) was studied for 3 minutes and was expressed asunits per minute per mg protein.

In situ hybridization

In situ hybridization was performed according to themethod described by Wilkinson (’93) with modifica-tions. Paraffin embedded longitudinal embryonic sec-tions were rinsed twice in 0.1M PBS, fixed in 4% para-formaldehyde in PBS for 10 min. at 4°C, and stored in70% ethanol at 220°C until use. For in situ hybridiza-tion 33-mer antisense oligonucleotide probes for ratCu/Zn SOD (nucleotides from 88 to 120) were synthe-sized by 394 Synthesis Setup Listing (Version 2.01).Sense probes were also synthesized and were used fornegative control. [35S] dATP labeled oligo-probes wereprepared using the DNA 39-end Labeling System (Pro-mega, Madison, USA). The probes were purified usingBio-Spin 6 Chromatography Columns (Bio-Rad, Her-cules, CA, USA). The specific activity was 4–5 3 109

cpm/mg. The slides containing the sections were re-hydrated for 10 min in 0.1M PBS, pre-treated with

proteinase K 10 mg/ml, diluted in 0.1 M Tris buffer, pH8.0, 50mM EDTA, pH 8.0 (Sigma) at 37°C for 10 min.

The coverslips were subsequently re-fixed in 4%paraformaldehyde, acetylated with 0.25% acetic anhy-drite in 0.1 M triethanolamine (Sigma), and rinsed in2 3 SSC (10%) for 10 min. The slides were hybridizedin a solution containing 50% formamide, 4 3 SSC,(20%) 10% dextran sulfate, 10 mM DTT, 500 mg/mltRNA, 13 Denhardt’s, and 100 mg/ml ssDNA (all fromSigma) in a humidified slide box for overnight at 37°C.100% SSC (or 20 3 SSC) is composed of 3M NaCl, 0.3M sodium citrate adjusted to pH 7.0 with 1 ml/L ofdiethylpyrocarbonate (DEPC). 30 ml hybridizationbuffer was added to each cover slip, which containedthe probe with a specific activity of 1 3 106 cpm/mg. Allthe solutions used before the process of washing theslides were pre-treated with 0.1% diethyl-pyrocarbon-ate (Sigma) to destroy RNase activity. The slides werethen washed with SSC containing 0.1 M mercapto-ethanol as follows: 2 3 SSC for 10 min at room tem-perature; 0.2 3 SSC twice for 30 min at 50°C; 0.1 3SSC twice for 30 min at 52°C; 1 3 SSC for 10 min atroom temperature. Finally, the cover slips were airdried and then mounted face up on slides. The slideswere dipped in NTB-2 emulsion (Eastman Kodak,Rochester, NY) diluted 1:1 with water, dried at roomtemperature for overnight, and exposed at 4°C in des-iccated slide boxes for 3 and 4 weeks. The exposedslides were developed in D-19 developer at 24°C for 4min, fixed in Unifix for 5 min, and finally washed withwater for 10 min. Slides were counter stained withHematoxilin and mounted using ENTELLAN (Merck,Germany). The results were assessed evaluating theintensity and location of the silver grains in the sec-tions or individual cells. The whole embryonic sectionwas examined by a low magnification 340. By 1003magnification the organs were identified. Intensity ofthe grains was measured on a scale of 1–4, 4 being thehighest intensity. By 10003 magnification the deposi-tion of grains in the embryonic cells was examined andthe average number of positive cells from 300–350cells/field was calculated. All observations and photo-

TABLE 1. Average reducing power of water-soluble antioxidants as reflected by the anodic current and peakpotential in 10.5-day-old rat embryos or yolk sacs cultured for 28 h in diabetic serum with and without

vitamins C and E compared with controls

Treatment groupNo. of

embryos

Sac Wave I embryo Wave II embryo

Peak potent(mV)

Anodic current(mA)

Peak potent(mV)

Anodic current(mA)

Peak potent(mV)

Anodic current(mA)

Diabetes 50 675 6 8.15a 1.48 6 0.18b 392 6 10.57 2.08 6 0.11b No wave No waveDiabetes vitamins

C and E 30 697 6 11.78 2.52 6 0.08 382 6 6.24 3.50 6 0.05 785 6 5.53 4.18 6 0.17Control 20 692 6 12 2.65 6 0.1 384 6 11 3.52 6 0.1 781 6 14 4.25 6 0.2Control vitamins

C and E 50 698 6 12.05 2.85 6 0.20 395 6 9.72 3.68 6 0.08 793 6 7.26 4.50 6 0.12†Peak potential was measured as half-increase of the anodic current at each wave.aMean 6 SE.bSignificantly lower than all other groups (ANOVA and Bonferroni t-test); P , 0.05.cSignificantly lower than control with and without vitamins C and E (ANOVA and Bonferroni t-test); P , 0.05.

36 ZAKEN ET AL.

Fig. 1. Representative cyclic voltammogram (CV) of water-soluble fraction in a 10.5 day-old rat embryo cultured for 28hin: A-normal serum; B-“diabetic” serum; C-“diabetic” serumwith vitamins C and E. Arrows indicate the first and secondanodic waves (peak potentials). Note that the second peakpotential of the control embryo is missing in the “diabetic”embryo and reappears in “diabetic” embryo with vitamins Cand E.


graphs were made on a Zeiss photomicroscope (Zeiss,Germany) using Kodak Ektar 100 film.

Statistical analysis

This was performed using Anova and t test withBonferroni’s adjustment and Fisher’s exact-test. Sig-nificance was set at P , 0.05.

RESULTS

The results of cyclic voltammetry are presented inTable 1 and Figure 1. In the embryos cultured in nor-mal rat serum and in those cultured in rat serum withvitamins C and E (both control groups) two peak po-tentials were found: wave I and wave II. Under diabeticconditions only one peak potential was found implyingthat an entire group of LMWA is either very low ormissing. The concentrations of water-soluble LMWA(as revealed by the size of the anodic current) werereduced in the embryos and yolk sacs under diabeticconditions as compared to embryos cultured in normalserum or in serum with vitamins C and E: anodiccurrent 1.48 6 0.18 mA vs. 2.60 6 0.07 and 2.85 6 0.20mA in sacs and 2.08 6 0.11 mA vs. 3.52 6 0.10 and3.68 6 0.08 mA in wave I of the embryos. Following theaddition of vitamins C and E to the “diabetic” culturemedium, the concentrations of LMWA in the embryosand yolk sacs returned to normal and the second peakpotential reappeared in the embryos (Figure 1 andTable 1). There was only one peak potential in thecontrol as well as diabetic yolk sacs.

HPLC studies of the embryos and yolk sacs haveshown a reduction in the concentrations of vitamins Cand E and an increase in uric acid under diabeticconditions in comparison to both control groups: Vit. C5.75 6 0.21 vs. 13.48 6 0.31 mM/mg protein in embryos(Table 2); Vit. E 10.18 6 0.98 vs. 90.79 6 0.87 mM/mgprotein (Table 3); Uric acid 16.95 6 0.21 vs. 1.90 6 0.09mM/mg protein (Table 4). Uric acid was not detected incontrol embryos with vitamins C and E (Table 4). Fol-lowing the addition of vitamins C and E to the diabeticculture medium, the level of vitamins C and E in theembryos and yolk sacs returned to normal and the levelof uric acid decreased in comparison to diabetics (Ta-bles 2–4).

SOD and CAT activity were decreased in the em-bryos and yolk sacs cultured in diabetic culture me-dium in comparison to controls, when calculated perembryo and yolk sac or per mg protein. Following theaddition of vitamins C and E to the “diabetic” mediumtheir activity in the embryos and yolk sacs returned tonormal (Tables 5, 6). The results of CV, the concentra-tions of LMWA, SOD and CAT activity in embryoscultured in control rat serum without vitamins C and Ewere also described elsewhere (Ornoy et al., ’99).

In situ hybridization for SOD mRNA in the longitu-dinal sections of the control embryos showed, underlow magnifications, an intensive deposition of grains(SOD mRNA transcripts) in the various organs (Fig. 2).The highest intensity of grains (SOD mRNA) was ob-served in the brain, spinal cord and heart. Under highmagnifications, more than 50% of the cells in the var-

TABLE 2. Concentration of vitamin c in 10.5-day-old-rat embryos and yolk sacs cultured for 28 h in diabeticserum with and without vitamins C and E compared with controls


embryos

Vitamin C concn

Sac Embryo

mM/sac mM/mg prot mM/emb mM/mg prot

Diabetes 40 0.72 6 0.05a,b 4.81 6 0.41b 1.18 6 0.04b 5.75 6 0.21b

Diabetes vitamins C and E 20 2.48 6 0.07c 12.08 6 0.23c 3.18 6 0.03c 10.61 6 0.09c

Control 20 3.075 6 0.07 13.98 6 0.32 3.77 6 8.08 12.15 6 0.25Control vitamins C and E 20 3.42 6 0.05 14.86 6 0.18 4.45 6 0.10 13.48 6 0.31aMean 6 SE.bSignificantly lower than all other groups (ANOVA and Bonferroni t-test); P , 0.05.cSignificantly lower than control with and without vitamins C and E (ANOVA and Bonferroni t-test); P , 0.05.

TABLE 3. Concentration of vitamin E in 10.5-day-old rat embryos and yolk sacs cultured for 28 h in diabeticserum with and without vitamins C and E compared with controls


embryos

Vitamin E concn

Sac Embryo


Diabetes 40 1.25 6 0.06a,b 8.33 6 0.85b 2.24 6 0.07b 10.18 6 0.98b



38 ZAKEN ET AL.

ious organs of the control embryos were positive (Table7–10). The SOD mRNA transcripts in the embryonicsections were significantly reduced in all organs of“diabetic” embryos in comparison to controls (Tables7–10, Fig. 3). Addition of vitamins C and E to “diabetic”medium abolished the decrease of the expression ofSOD mRNA in all organs examined, but it was stilllower than in the embryos cultured in control serumwith vitamins C and E. The SOD mRNA transcripts insections of embryos cultured in control rat serum withoutVitamins C and E were similar to those cultured in con-trol serum with vitamins C and E (Tables 7–10, Fig. 4).

DISCUSSION

The culture of 10.5 day-old rat embryos in rat serumcontaining high concentrations of glucose and ketone

bodies decreased the activity of SOD and CAT in em-bryos and yolk sacs and the concentration of LMWA.HPLC studies showed a reduction of ascorbic acid andof vitamin E in experimental embryos and yolk sacswith a concomitant increase in uric acid content. Thesechanges were almost completely reversed by the addi-tion of vitamins C and E to the diabetic culture me-dium. The addition of these vitamins to normal ratserum had no effect on the embryos. We found previ-ously (Ornoy et al., ’99) that the impairment of growthand reduced protein content of rat embryos under “di-abetic” culture conditions was corrected by the additionof vitamins C and E to the culture medium. In thepresent study we showed that the protective effects ofthese two antioxidants were exerted by the improve-ment of the embryonic antioxidant defense mechanism.

TABLE 4. Concentration of uric acid in 10.5-day-old rat embryos and yolk sacs cultured for 28 h in diabeticserum with and without vitamins C and E compared with controls


embryos

Uric acid concn

Sac Embryo


Diabetes 40 4.28 6 0.07a,b 28.53 6 0.48b 3.32 6 0.04b 16.95 6 0.21b


Control 20 1.30 6 0.1 5.91 6 0.45 0.59 6 0.03 1.90 6 0.09Control vitamins C and E 20 ND ND ND ND

ND, not detected.aMean 6 SE.bSignificantly higher than all other groups (ANOVA and Bonferroni t-test); P , 0.05.cSignificantly higher than control with and without vitamins C and E (ANOVA and Bonferroni t-test); P , 0.05.

TABLE 5. Activity of SOD in 10.5-day-old rat embryos and yolk sacs cultured for 28 h in diabetic serum withand without vitamins C and E compared with controls


embryos

SOD concn

Sac Embryo

U/min/sacU/MIN/mg

prot U/min/embU/min/mg

prot

Diabetes 50 0.21 6 0.02a,b 1.43 6 0.01b 0.33 6 0.02b 1.53 6 0.04b

Diabetes vitamins C and E 30 0.42 6 0.01c 2.23 6 0.03 0.70 6 0.02 2.35 6 0.06Control 20 0.51 6 0.01 2.32 6 0.04 0.75 6 0.02 2.42 6 0.06Control vitamins C and E 50 0.54 6 0.02 2.39 6 0.04 0.81 6 0.03 2.62 6 0.05aMean 6 SE.bSignificantly lower than all other groups (ANOVA and Bonferroni t-test); P , 0.05.cSignificantly lower than control with and without vitamins C and E (ANOVA and Bonferroni t-test); P , 0.05.

TABLE 6. Activity of CAT in 10.5-day-old rat embryos and yolk sacs cultured for 28 h in diabetic serum withand without vitamins C and E compared with controls


embryos

CAT concn

Sac Embryo

U/min/sac U/min/mg prot U/min/embryo U/min/mg/prot

Diabetes 50 3.50 6 0.15b 23.33 6 0.80b 4.69 6 0.17b 21.70 6 0.17b




The addition of vitamin C and vitamin E to the diabeticculture medium increased the concentrations of thesetwo antioxidants in the embryos and yolk sacs, improv-ing embryonic antioxidant defense mechanisms. Vita-mins C and E that are able to cross the placenta (Choiand Rose, ’89; Mino and Nishino, ’73), protected theembryos in the same way as they protect oxidativestress damage in tissues of adults (Carr et al., ’00;McCall & Frei, ’00; Trevithick et al., ’89). We shouldremember that the mechanism for ROS inactivation byLMWA and by the antioxidant enzymes differs. SODradicals forming hydrogen peroxide; CAT and GSH-Px,as well as other CAT like enzymes, decompose hydro-gen peroxide (Halliwell and Gutteridge, ’95), thus act-ing as reducing antioxidant scavengers.

Uric acid, which is produced in the cells as wasteproduct, is also known to be increased during oxidativestress and tissue damage, especially cell necrosis(Stower et al., ’97; Ornoy et al., ’99). Increased embry-onic neural tube cell death under diabetic conditions

was indeed observed by Phelan et al. (’97). Increasedapoptosis explains the high concentrations of uric acidin embryos cultured under diabetic conditions. Treat-ment with antioxidants reduces embryonic damageand hence the uric acid contents.

Uric acid is detected in the CV at the first anodicwave (AW), at peak potential of 300–400 mV. How-ever, in the first AW other water-soluble antioxidantscan also be found. In spite of the increase in uric acidlevels in diabetic conditions the first AW is lower thancontrols. This can be explained by the fact that otherantioxidants which appear at the AW (i.e. vitamin C orNADH) are decreased, and the total concentration ofantioxidants are still lower than in controls.

In the control embryos, under normal condition ofembryonic growth, the level of uric acid is very low. Itis further decreased in controls following the additionof vitamins C and E to undetectable levels. Althoughwe have no explanation to that fact, it may imply thatunder such conditions embryonic oxidative stress is

TABLE 7. SOD mRNA expression in the brain of 10.5-day-old rat embryos cultured for 28 h in diabetic

serum with and without vitamins C and E comparedwith controls

Treatment group

Score from 1–4of positive

cells (3100)

No. and % of positivecells from 300–350/field

(31,000) %

Diabetes 1.48 6 0.06a,c 48.21 6 2.08b,c 14.83c

Diabetes vitaminsC and E 3.37 6 0.05d 170.31 6 2.91d 52.4

Control 3.61 6 0.06 179.99 6 2.99 55.38Control vitamins

C and E 3.76 6 0.04 192.86 6 3.48 59.34aMean 6 SE intensity of the grains as measured on a scale of1–4 with the highest intensity.bMean 6 SE number of positive cells/field.cSignificantly lower than all other groups (ANOVA and Bon-ferroni t-tests); P , 0.05.dSignificantly lower than controls; P , 0.05.

TABLE 8. SOD mRNA expression in the spinal cordof 10.5-day-old rat embryos cultures for 28 h in

diabetic serum with and without vitamins C and Ecompared with controls

Treatment group


cells (3100)

No. and % of positivecells from

300–350/field

(31,000) %

Diabetes 1.52 6 0.05a,c 50.62 6 2.36b,c 15.57Diabetes vitamins

C and E 3.39 6 0.05d 159.36 6 2.53d 49.03Control 3.50 6 0.06 171.51 6 2.80 52.77Control vitamins

C and E 3.65 6 0.04 181.25 6 3.74 55.77aMean 6 SE intensity of the grains as measured on a scale of1–4, with 4 the highest intensity.bMean 6 SE number of positive cells/field.cSignificantly lower than all other groups (ANOVA and Bon-ferroni t-test); P , 0.05.dSignificantly lower than controls (ANOVA and Bonferronit-test); P , 0.05.

TABLE 9. SOD mRNA expression in the heart of 10.5-day-old rat embryos cultured for 28 h in diabetic


Treatment group


cells (3100)


300–350/field

(31,000) %

Diabetes 1.51 6 0.05a,c 50.31 6 1.86b,c 15.48Diabetes vitamins

C and E 3.50 6 0.06d 171.43 6 2.01d 52.75Control 3.68 6 0.05 190.97 6 2.98 58.96Control vitamins

C and E 3.72 6 0.05 191.77 6 3.53 59.01aMean 6 SE intensity of the grains as measured on a scale of1–4, with 4 the highest intensity.bMean 6 SE number of positive cells/field.cSignificantly lower than all other groups (ANOVA and Bon-ferroni t-test); P , 0.05.dSignificantly lower than controls (ANOVA and Bonferronit-test); P , 0.05.

TABLE 10. SOD mRNA expression in the liver of 10.5-day-old rat embryos cultured for 28 h in diabetic


Treatment group


cells (3100)


300–350/field

(31,000) %

Diabetes 1.42 6 0.05a,b 45.06 6 2.00b,c 13.86Diabetes vitamins

C and E 3.18 6 0.05 166.26 6 3.13 51.16Control 3.37 6 0.06 172.00 6 2.32 52.92Control vitamins

C and E 3.39 6 0.05 176.03 6 3.57 54.16aMean 6 SE intensity of the grains as measured on a scale of1–4, with 4 the highest intensity.bMean 6 SE number of positive cells/field.cSignificantly lower than all other groups (ANOVA and Bon-ferroni t-test); P , 0.05.

40 ZAKEN ET AL.

negligible and embryonic growth is optimal. It may bethat vitamin C and/or vitamin E serve as protectingantioxidants and the damage to the cell is prevented bytheir excess, arresting the production of uric acid. Thehigh levels of uric acid in “diabetic” embryos do notseem to protect the embryo from oxidative damage asuric acid is produced, at least in part, secondary to thecell and tissue damage induced by the diabetic envi-ronment. Moreover, in high concentrations uric acidmay even serve as a pro-oxidant, as in high concentra-tions it can delay redox recycling of a tocopherol caus-ing its depletion and promoting lipid peroxidation(Benzie & Strain, ’96). It is also possible that as a resultof diabetes-induces oxidative stress the cells respondby up-regulation of the uric acid system. We shouldalso remember that these results might also point to anadditional mechanism/s of diabetes-induced embryonicdamage.

Of the lipid soluble antioxidants, vitamin A and caro-tenoids as well as vitamin E seem to play an importantrole preventing lipid peroxidation by donating hydro-gen to peroxil (ROO2) radicals and stopping the chainreaction (Sharma & Buetner, ’93). They are often used

as protective agents against oxidative stress in somechronic diseases (Olson, ’96). Carotenoids can serve asan electron acceptor and as electron donor. Vitamins A,E and C may induce inhibition of cell growth and en-hance cell differentiation, thus improving the efficacyof cancer therapy (Prasad et al., ’99). We found a re-duction of vitamin E under diabetic conditions, imply-ing that the diabetic environment might increase in theembryo lipid peroxidation, that is apparently inhibitedby the addition of vitamin C and E. These vitamins,when used in clinical practice, were found to be helpfulin the prevention of a variety of chronic diseases suchas cardiovascular diseases, cataract asthma and pre-eclampsia (Chapell et al., ’99; McDermott, ’00). Theymay as well be used for the prevention of diabetes-induced embryonic damage.

A reduction of antioxidant defense in diabetic cultureconditions was found in the yolk sacs as well as in theembryos, pointing to a possible involvement of the yolksac in the diabetes-induced embryonic damage. In ourprevious studies we observed specific morphologicaldamage to the yolk sacs of 10.5 day old rat embryoscultured in diabetic medium (Zusman et al., ’87). In

Fig. 2. Photomicrograph of a section throughthe brain of a control rat embryo to show themassive expression of SOD mRNA (many posi-tive grains). 3600 [Color figure can be viewed inthe online issue, which is available at www.interscience.wiley.com.]


addition we also found decreased transfer of sucrose(reduced endocytic index) in yolk sacs of rat embryoscultured in serum from women with poorly treatedpregestational or gestational diabetes (Ornoy et al.,’95). It is therefore possible that compromised ability ofthe yolk sac to transport required substances to theembryo in diabetic conditions might damage the devel-oping embryo and also reduce embryonic antioxidantdefense capacity. This can be further studied by addi-tional elaboration of the transport and synthetic func-tions of the yolk sac in diabetes and its specific impacton various synthetic pathways in the embryo.

A reduction of embryonic SOD mRNA under diabeticconditions, as revealed by in situ hybridization mayresult from a direct effect of the diabetic metabolicfactors on the SOD genome. If this is the major expla-nation, it is difficult to understand why vitamins C andE reduce that suppressive effect of diabetes on SODgene, as those vitamins are not expected to affectgenomic functions (Bendich & Langseth, ’95; Halliwelland Gutteridge, ’95). There may be, however, severalalternative explanations:

1) Diabetic conditions affect gene transcription. Inthat context it is important to stress that Cederberg etal. (’00) found in embryos of diabetic H rats, that areresistant to diabetes-induced teratogenicity, increasedmRNA levels of Mn-SOD and catalase. However, theydid not find any difference in the DNA sequence of thepromotor region between these rats and the U rats thatare susceptible to diabetes-induced anomalies. They

explained their findings by differences in transcrip-tional regulation (Cederberg et al., ’00). Thus, diabeticenvironment may decrease antioxidant gene expres-sion by affecting transcriptional factors.

2) The increased ROS produced under diabetic con-ditions and not the diabetic metabolic factors affectSOD gene expression and possibly the expression ofgenes of other antioxidant enzymes. The added vita-mins C and E reduce embryonic oxidative stress, andhence the effects of the diabetic environment on SODgene are abolished. This possibility of superoxide rad-icals-induced altered gene expression also explains thenormal activity of SOD and CAT when these LMWAwere added to the diabetic culture medium.

3) It is possible that under diabetic conditions thereis a depletion of the antioxidant enzymes as a result ofthe “exhaustion” of these enzymes while the embryo istrying to cope with the oxidative stress. This is ex-pected, in turn, to increase SOD mRNA, which is notincreased because of the suppressive effects of the di-abetic environment on the SOD genome. Under suchconditions, the added vitamin C and E reduce the oxi-dative stress decreasing the need for heightened activ-ity of these enzymes.

We should remember that our study was performedin culture, and the response of cultured embryos maybe different than in vivo, especially as maternal metab-olism may modify the protective effects of antioxidants.However, most of our previous studies on embryos in“diabetic environments” regarding yolk sac damage

Fig. 3. Photomicrograph of a sectionthrough the brain of a rat embryo cul-tured in diabetic serum to show reducedexpression of SOD mRNA. 3600 [Colorfigure can be viewed in the online issue,which is available at www.interscience.wiley.com.]

42 ZAKEN ET AL.

and congenital anomalies, showed similar results invivo and in vitro (Zusman et al., ’87). Moreover, anti-oxidants are known to have protective effects in vivoand in vitro. The concentrations of vitamins C and Eused by us are similar to the physiological concentra-tions. It is therefore reasonable to presume that theprotective effects of antioxidants on diabetes-inducedembryonic damage will be evident also in vivo, andapparently in species other than rats.

In conclusion. Vitamins C and E abolished thedamaging effects of the diabetic environment whenadded to the culture medium in rather low concentra-tions. It seems, therefore, that a clinical trial in dia-betic pregnant women to assess the possible protectiveeffects of vitamins C and E on the human fetus seemsto be of primary importance, as these two vitamins arein routine clinical use. Indeed, such a clinical trial(approved by Helsinki Committees) is now carried outby us in Israel.

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vitamins c and e improve rat embryonic antioxidant defense mechanism in diabetic culture medium

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