Ali S. H. Alchalabi*, Erkihun Aklilu, Abd Rahman Aziz, F. Malek, S. H. Ronald, Mohd Azam KhanFaculty of Veterinary Medicine, UMK City Campus, Pengkalan Chepa, Locked Bag36, 600 Kota Bharu, Kelantan Mala ysiaSchool of Electrical System Engineering, Universiti Malaysia Perlis (UniMAP), Pauh Putra, 0600 Arau, Perlis Malaysia

ABSTRACT

Objective:To assess the intterine irradiation of 1800 MHz Global System of Mobile telecommunication on pre- and postnatal development in Sprague-Dawley rats. Methods:The whole-body irradiation 1 hr./ day and 2 hr./day was applied to the pregnant rats in three different intervals (one week, two weeks and three weeks) at SAR 0.048 W/Kg and control groups. Post-Morton findings and growth markers were monitored. Sera were collected for biochemical analysis. Results:Prenatal development findings showed uterine congestion, haemorrhage, dead and reabsorbed fetuses were observed in exposure groups during 2nd and 3rd week of pregnancy unlike to control. 1st and 2nd week in-utero irradiation showed significant reduction with unequal and asymmetrical distribution of implantation sites and embryos in exposure groups except the control group. A number of live embryos were significantly reduced with an increasing number of dead and reabsorbed embryos in the 2 h/day of the 2 nd-week exposure group compared to control group. Malformation, haematoma, and oedematous foetuses in experimental groups were observed unlike control foetuses. A significant decrease in live foetuses and a significant decrease in body mass of foetuses at gestation day 20, unlike control group. Postnatal observations showed haematoma, congestion, short tail, malformation and growth restriction and delay in some growth markers were observed. In-utero irradiation for 2 and three weeks induced oxidative stress in pregnant rats. Conclusion:Results suggest that long-term exposure to EMF during the pregnancy lead to chronic stress, which has detrimental effects on pre-& postnatal development and for that more studies to clarify such harmful effects are recommended.

ARTICLE INFO

Article history:

Received 29 August 2015

Received in revised form 16 October 2015

Accepted 10 November 2015

Available online 1 January 2016

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Different periods of intrauterine exposure to electromagnetic field:Influence on female rats’fertility, prenatal and postnatal development

Ali S. H. Alchalabi1*, Erkihun Aklilu1, Abd Rahman Aziz1, F. Malek2, S. H. Ronald2, Mohd Azam Khan1
1Faculty of Veterinary Medicine, UMK City Campus, Pengkalan Chepa, Locked Bag36, 16100 Kota Bharu, Kelantan Mala ysia
2School of Electrical System Engineering, Universiti Malaysia Perlis (UniMAP), Pauh Putra, 02600 Arau, Perlis Malaysia

ABSTRACT

Objective:To assess the intterine irradiation of 1800 MHz Global System of Mobile telecommunication on pre- and postnatal development in Sprague-Dawley rats. Methods:The whole-body irradiation 1 hr./ day and 2 hr./day was applied to the pregnant rats in three different intervals (one week, two weeks and three weeks) at SAR 0.048 W/Kg and control groups. Post-Morton findings and growth markers were monitored. Sera were collected for biochemical analysis. Results:Prenatal development findings showed uterine congestion, haemorrhage, dead and reabsorbed fetuses were observed in exposure groups during 2nd and 3rd week of pregnancy unlike to control. 1st and 2nd week in-utero irradiation showed significant reduction with unequal and asymmetrical distribution of implantation sites and embryos in exposure groups except the control group. A number of live embryos were significantly reduced with an increasing number of dead and reabsorbed embryos in the 2 h/day of the 2 nd-week exposure group compared to control group. Malformation, haematoma, and oedematous foetuses in experimental groups were observed unlike control foetuses. A significant decrease in live foetuses and a significant decrease in body mass of foetuses at gestation day 20, unlike control group. Postnatal observations showed haematoma, congestion, short tail, malformation and growth restriction and delay in some growth markers were observed. In-utero irradiation for 2 and three weeks induced oxidative stress in pregnant rats. Conclusion:Results suggest that long-term exposure to EMF during the pregnancy lead to chronic stress, which has detrimental effects on pre-& postnatal development and for that more studies to clarify such harmful effects are recommended.

ARTICLE INFO

Article history:

Received 29 August 2015

Received in revised form 16 October 2015

Accepted 10 November 2015

Available online 1 January 2016

Keywords:

Tel:+60112947731

E-mail:alisaeedchalaby@yahoo.com

Foundation priject:The project was fully supported by Faculty of Veterinary Medicine of Universiti Malaysia Kelantan.

1. Introduction

Potential risk of radio-frequency electromagnetic wave (RFEMW) emitted by modern mobile phone communications technology for human environment, and health is strictly connected to contemporary approaches to assign safety limits for cell phones, wireless fidelity (Wi-Fi) as well as all electronic devices operate within microwave range. Safety limits that defined by International Commission of Non-Ionizing Radiation Protection (ICNIRP) guidelines based on the thermal effect of short-term exposure to non-ionizing radiation within microwave range on the biological system[1]. Nowadays, a huge amount of research articles of epidemiological, human, animal, cellular, mechanisms and dosimetry studies points to non-thermal effect of microwave (MW) on the biological system after chronic exposure of the enviroment and human population to RF-EMW (heavy use of mobile phone communication devices)[2-5]. The latest reseach agenda of World Health Organization (WHO) on Radiofrequency fields (RF) has prompted studies on different ages of children in addition to animal work to investigate RF exposure effect on prenatal development and behavior and viewing the effect of RF on early life stages[6].Electromagnetic fields (EMF) within frequency 900-1800 MHz Global System of Mobile telecommunication (GSM) and 2450 MHz of Wi-Fi signals have increased public concern as to health effect with exclusive attention to the adolescent population. ICNIRP and Institute of Electrical and Electronics Engineers (IEEE) do not involve pregnant women and their babies in their safety limit levels of RF-EMW exposure, although they involve workers and general public in their limit levels of RF-EMW[1,7]. WHO recently reported that ‘the accumulated evidence did not establish the existence of adverse short or long-term health effects from the signals produced by base stations and local wireless networks’ and the ‘mechanistic understanding would address the possibility that children may react to RF more effectively than adults” [8]. In the last 10 years, several articles have been published showing the effect of radio-frequency radiation (RFR) intrauterine exposure on dams and their newborns. These studies investigated the effect of RFR on oxidative stress, DNA damage, teratogenicity, neurodevelopment, embryogenesis and behavior in laboratory animals and avian species[5,9-12]. They suggest that the RFR & GSM-like signals had adverse effects on pregnant dams as well as their newborns and had detrimental effects on avian embryos after exposure either to mobile phone or signal generator within frequency of 850 MHz -1800 MHz. On the other hand, a few reported contradictory results regarding the effect of intrauterine Wi-Fi exposure of pregnant rats and postnatal development. Teratology and development studies have not detected any noxious effects of exposure to mobile phone-related RF fields at exposure levels below standard levels[13]. Contradictory study in Turky found growth restriction and delayed puberty in female Wister rats due to intrauterine and early life stage exposure to Wi-Fi signal[2]. Long term “heavy use” exposure to mobile phone communications technology either via mobile phones or via mobile phone base stations radiation which is the main and important sources of RFR in our environment during different stages of pregnancy should be considered as a serious problem that needs to be addressed adequately. The lack of solid and unwavering conclusive study that establishes the deleterious effect of electromagnetic radaitions from mobile phones and base stations on pregnant women, and newborns justifies the need for further and extensive studies in this regard. Therefore, this study was designed to investigate the bio-effects of 1800 MHz GSM-like RF-EMF of mobile phone on different periods of pregnancy on female rats’ fertility, prenatal and postnatal development of pups.

2. Material and methods

2.1. Animals and study desgin

The study was approved by the scientific committee of Faculty Veterinary Medicine of University Malaysia Kelantan (UMK) and was conducted in accordance with the UMK guidelines for animal experiments (FPV-PGSC-2014). Sprague- Dawley rats were bred at room temperature (24±10) ℃ and humidity of (60 ± 10)% (relative humidity) with light / dark cycle 12-12 hour in the laboratory animal research unit of Faculty of Veterinary Medicine, (UMK), tap water and standard rat pellet were provided ad libitum. For intrauterine exposure in different periods, gestation period was divided into three intervals 1st week, 2nd week and 3rd week.

Sixty virgin female rats (in each interval), 12 weeks of age at the start of experiment were used. Females were mated with unexposed adult fertile male rats of the same strain in a 1:2 ratio (male:female) for a maximum of 15 nights. During the cohabitation period, vaginal swab samples were examined microscopically every morning for the presence of sperm. The day of finding a copulatory plug or sperm was considered as gestation day 0. All females were observed daily for mortality and for physical signs from initiation of exposure. Animals were kept in Plexiglas cages. Especially designed exposure Plexiglas box (60 cm × 40 cm × 20 cm) was used during the exposure time because Plexiglas is non-conductive material that is not affected by RF-EMR. The protocol of RF-EMR exposure was done as follows:

a) 1st-week interval exposure was started from Gestation Day(GD) 0 until GD 7; b) 2nd-week interval exposure was started from GD 0 until GD 14; c) 3rd-week interval exposure was started from GD 0 until GD20 one day before delivery.

Each experimental interval was composed of:1) Control group (n=20); 2) 1h/day exposure group (n=20); 3) 2h/day exposure group (n=20).

At the end of each interval of the experiment, the experimental animals were divided into two subgroups; A and B. Female fertility and prenatal development were evaluated in each interval of subgroup’s A females, whereas postnatal development was evaluated in pups of subgroup’s B females.

2.2. Radiofrequency electromagnetic radiation setup.

Pregnant dams were randomly distributed into nine groups (three groups/interval and 20 animals/group) SAR level 0.048 W/kg was calculated using the equation:

Where (E) is the magnitude of electric field 38.63 v/m, (σ) is the conductivity 1.34 s/m and (P) is the mass density of the tissueequivalent media 1090 kg/m3[14]. Whole-body radiofrequency radiation (RFR) exposures were done. (Figure 1).

Figure 1.　Showing the EMF exposure setup using GSM like radiofrequency generator system.

GSM-like signals at a frequency of 1 800 MHz were provided by a signal generator (Agilent Technologies E8267D, 250KHz - 20GHz PSG Vector Signal Generator) with an integrated pulse modulation unit and horn antenna (A-INFOMW Standard Gain Horn Antenna 1.7 - 2.6 GHz WR430, China) in an exposure room. The signals were amplitude-modulated by rectangular pulses with a repetition frequency of 217 Hz and a duty cycle of 1:8 (pulse width 0.576 msec), corresponding to the dominant modulation component of the GSM. The RFR generator provided 20 dBm (0.1W) powers during the exposure period[15-17].

2.3. Maternal observations

2.3.1. Body weights

All animals were weighed individually on time zero ( one day before starting the experiment) and at the end each interval of an experiment by using a balance with 0.01 g sensitivity.

2.3.2. Clinical observation

Daily clinical examination was performed on females during experimental durations for clinical signs:abortion, mucous membrane status, behavior and nervous signs.

2.4. Fertility and prenatal development

At the end of an exposure period of each interval, animals in subgroups A anesthetized by chloroform and all females were euthanized on GD7, 14 and 20, cesarean section were done. The uterus was removed, opened and pregnancy status recorded. During the 1st-week interval, the uterus of each dam was examined to determine the number of implantation sites and number of blastocysts in each uterine horn. During 2nd week interval, the uterine horns were examined to determine (1) the number of live or dead fetuses per uterine horn, (2) the number of early and late resorption sites on each uterine horn. While in duration, 3rd week of pregnancy at GD 20 total number of fetuses, number of alive, dead fetuses and fetuses were weighed individually. Macroscopic abnormalities were checked carefully.

2.5. Postnatal development

Females of subgroup B after finishing RF-EMR exposure for all three intervals of pregnancy were observed carefully until natural delivery happened. The pregnant females were separated individually in monitoring cages from delivery day until 30 days.

2.5.1. Delivery data

Each dam was examined after 24th hour of natural delivery to count the total numbers of pups/ litter /group / interval, alive, dead and with anomalies.

2.5.2. Body weight of pups

Pups/ litter /group / intervals were weighed on 24 h. after delivery, D7, D14, D21, and D28 individually (30 pups / group were taken for weekly weight gain).

2.5.3. Normal physiological development

Following a general examination, the number of pups/litter/group /interval monitoring for the following physiological development. 30 pups/group/interval were checked carefully and individually for following parameters:Ear appearance, hair appearance, teeth appearance, eye opening and weaning day as an indicator for normal growth.

2.6. Biochemical analysis

A rapid method for measuring malondialdehyde (MDA) as a bio-marker of lipid peroxidation in plasma samples was done, which based on the thiobarbituric acid (TBA) test by use of spectrophotometric quantification of pink MDA-TBA complex formed by reaction of MDA with two molecules of TBA. Lipid Peroxidation (MDA) Assay Kits (ab118970, abcam ) were used according to the manufacturer’s instructions. Briefly 10 μL plasma with 500 μL of 42 mM H2SO4in a micro centrifuge tube was added. Add 125 μL of phosphotungstic acid solution and mix by vortexing. Incubation at room temperature for five min., after that centrifuge for three min. at13 000 × g. The pellets were collected and resuspended on ice with 100 μL ddH2O [with 2 μL BHT (100×)]. The final volume was adjusted to 200 μL with ddH2O, and then 600 μL of TBA solution into each tube containing standard and sample was added. Incubation at 95 ℃ for 60 min. Following reaction with TBA, MDA was directly quantified using spectrophotometer at wave length 532 nm[18].

Glutathione peroxidase (GSH-PX) concentration in serum was measured by using rat glutathione peroxidase Elisa kit (CUSABIORat GSH-PX ELISA Kit, CSB-E12146r, WuhanUniversity Science, Wuhan, Hubei province 430223, P.R.China). According to the manufacturer’s instructions, estimation was done.

Melatonin (MT) concentration in serum was estimated by using rat melatonin Elisa kit (CUSABIO MelatoninELISA Kit, CSBE13433r, Wuhan University Science, Wuhan, Hubei province 430223, P.R. China). Estimation was done according to the manufacturer’s instructions. Both GSH-PX and MT measurement were done as following procedure. Briefly, Reagents, samples and standard were prepared and 100 μL of samples and standard was added to wells and incubated 2 hr. at 37 ℃. Each well was removed from the contents and 100 μL of biotin- antibody was added and incubated for 1 hr. at 37 ℃. Washing three times with washing solution, 100 μL HRP-avidin was added to each well and incubated for 1hr. at 37 ℃. After that each well was washed 5 times by washing solution and 90 μL TMB substrate was added in each well, incubated for 20 min. and finally 50 μL stop solution was added to each well. Spectrophotometer was used at wave length at 450 nm for reading the assay plate.

2.7. Statistical analysis

All results were expressed as mean ± standard error (mean ± S.E.). Data were evaluated by using the statistical SPSS program v.19 software (SPSS In. Chicago, IL., USA). ONE and TWO-way ANOVA and LSD test was used to evaluate the significance between groups and P values of less than 0.05 were considered as significant.

3. Results

3.1. Dam’s body weight

ANOVA test for dams’ body weight revealed that there were no significant differences in mean body weight of the dams in experimental groups during 1st, 2nd and 3rd-week intervals.

Table 1　Effect of RF-EMR on dams’ body weight during a different period of pregnancy of experiment.

3.2. Clinical observations

Clinical examination showed rough skin was observed in some females in 2h/day exposure group of an interval 3rd-week experiment. Abortion and bleeding were observed in one female of 1h/day exposure group of the interval 3rd-week experiment. While no abnormal clinical signs were observed in other females in experimental groups of intervals 1st week and 2nd week. No death was noted in all intervals and groups of experiment. Post Morton (PM) findings showed uterine congestion, haemorrhage, dead and reabsorbed fetuses were observed in both exposure groups during 2nd and 3rd intervals of pregnancy, while no PM findings were observed in females of control groups (Figure 1)

Figure 1.　Interval 2nd week of pregnancy (A) control female shows a normal uterus with normal distribution of embryos in both uterine horns, (B, C) females’uterine horns of both exposure groups show congestion, haemorrhage, dead and reabsorbed embryos and were indicated by arrows, (D, E and F) females’uterine horns of both exposure groups show abnormal distribution and decrease in a number of embryos.

3.3. Fertility and prenatal development (laparotomy data) for subgroups A female

3.3.1. One-week exposure duration (GD 0-GD 7)

Number of implantation sites and number of blastocysts in the 2h/day exposure group were affected markedly with unequal and asymmetrical distribution of implantation sites in both uterine horns unlike the control group (Table 2) (Figure 2).

3.3.2. Two weeks exposure duration (GD 0-GD 14)

Two-week exposure to RF-EMR showed that the mean number of live embryos was significantly reduced, while a number of dead and reabsorbed embryos were increased significantly in the 2h/day exposure group in comparison with a control group (Table 2). 1h/day exposure to RF-EMR has no significant effect on a number of live, dead and reabsorbed embryos despite of there were some alterations in their values. Asymmetrical distribution of embryos was also observed in both uterine horns in both RF-EMR exposure groups except the control group (Figure 2).

3.3.3. Three-week exposure duration (GD 0-GD 20)

The uterine horns were affected significantly and exhibited some pathological signs such as congestion and haemorrhage in irradiated females of both exposure groups in comparison with a control group. Intrauterine exposure for three weeks affected negatively the prenatal development of foetuses represented by malformation, haematoma in different parts of the body, increase in skin thickness and oedematous foetuses (Figure 3). Intrauterine irradiation by 1800 MHz GSM-like signals in 1h/day and 2h/day groups exhibited a highly significant decrease in a mean total number of foetuses and number of alive foetuses in comparison with non-irradiated dams in a control group, while a number of dead foetuses remain consistent. Body mass of foetuses at GD20 was affected significantly in both irradiated groups (Table 2)

Figure 2.　Interval 2nd week of pregnancy. (A) Control normal uterus with normal distribution of embryos in both uterine horns, (B, C, D) early embryonic death was observed in RF-EMR exposure groups and indicated by arrows, (E, H) reabsorbed embryos were recorded in 1h/day exposure group, (F, G) early embryonic death was observed in RF-EMR exposure groups, (G, H, J) moderate to severe congestion was observed in uterine horns in all exposure groups. Unequal and asymmetrical embryonic distributions were very clear in (B, C, D, E, F, G, H, and J) unlike to (A) control uterine horns.

3.4. Postnatal development (Dealivery data) for subgroup’s B pregnant females

Exposure to RF-EMR in-utero during different intervals of pregnancy lead to different hazardous effects on live births and these effects include haematoma, rough skin, skin redness, congestion, short tail and malformation and manifestations of these signs depend on duration of intrauterine exposure (Figure 4).

3.5. Delivery data

Mean total number of newly born pups, number of live and dead pups in addition to a number of pups with anomalies after in-utero irradiation with 1800 MHz GSM-like RF-EMR during the 1st week of pregnancy were similar in all three groups. After two-week intrauterine exposure the delivery data exhibited a reduction in a total number of pups and number of live pups unlike control group pups. While dead and abnormal pups number similar to control pups. Total number of pups and live pups in exposure groups after three weeks of exposure were significantly reduced in comparison with control group and the number of dead and normal pups statistically remain normal (Table 3, 4 and 5).

Figure 3.　Interval 3rd week of pregnancy. (A) Control normal uterus with normal foetuses, (B, C) 1h/day and 2 h/day exposed rats’ uterus showed haemorrhage and congestion indicated by an arrow. (D, E, F) GD 20 foetuses showed haematoma, dead and oedematous foetuses in 1h/day exposed rats. (G, H, I) Sever haematoma with big size oedematous foetuses were observed in GD 20 of pregnancy in 2h/day 2h/day exposure group.

Table 2　Effect of RF-EMR on prenatal development during different periods of pregnancy.

Figure 4.　Intrauterine exposure to RF-EMR showed different hazardous effects. (A) Head haematoma with head deformity was observed in pup after exposure to 1h/day for three weeks. (B) Short tail, head deformity and haematoma on left foot and tail were observed in 1h/day exposure for three weeks. (C) left foot haematoma in pup after exposure to 1h/day irradiation. (D, E) haematoma on the base of the tail, on the lower jaw and skin redness with haematoma on the right arm after 2h/day intrauterine irradiation for three weeks. (F) congested pups with haematoma on the back and base of the tail on pup in the 2h/day exposure group.

Table 3　Effect of one-week intrauterine RF-EMR exposure on off-spring examination.

Table 4　Effect of two weeks intrauterine RF-EMR exposure on off-spring examination.

Table 5　Effect of three weeks RF-EMR intrauterine exposure on off-spring examination.

3.6. Pups weekly body weight gain

Mean body weight was taken for 30 pups/group/ week and measured weekly beginning from 24 hours after normal delivery until 28 days of age ( 24h, D7, D14, D21 and D28). Pup’s body weight after intrauterine exposure to RF-EMR during 1st week of pregnancy showed no effect and were similar in all experimental groups (Figure 5).

Figure 5.　Effect of RF-EMR intrauterine exposure for one week of pregnancy on weekly body weight gain of pups.

Body weights of pups aged 24 hours from in-utero irradiated dams for two weeks did not show significant difference compared with the control pups (P=0.551, P=0.335 1h/day and 2h/day exposure respectively). While the weekly body weight gains from D7-D 28 was significantly lower than control groups (P=0.001). Highly significant reduction in weekly body weight gain in new-born pups until D 28 (P=0.001) was observed in pups from in-utero irradiated dams for 20 days in both exposed groups compared with the control pups (Figure 6 and 7).

3.7. Normal physiological development

Postnatal normal physiological development was checked carefully and individually for 30 pups/group/interval for monitoring appearance of ear, hair, teeth, eye-opening and determining of weaning day. There were no remarkable differences among experimental groups and control group for postnatal physiological development after intrauterine exposure during 1st week of pregnancy (Figure 8). No difference was observed in ear appearancedevelopment after two-week of in-utero irradiation, 2h/day inutero irradiation hair appearance, eye opening development and weaning day took longer time than control pups, while after 1h/ day irradiation, the postnatal physiological development remains consistent (figure 9). Three weeks of in-utero irradiation effect on postnatal physiological development negatively by increased the days required for normal hair appearance and induced delay in teeth eruption in both irradiated groups. Eye opening took longer time to open normally than a control group after 2h/day exposure group and the weaning period showed an increase in length of weaning period in both in-utero irradiation groups in comparison to control group (Figure 10).

Figure 6.　Effect of RF-EMR intrauterine exposure for tow week of pregnancy on weekly body weight gain of pups.

Figure 7.　Effect of RF-EMR intrauterine exposure for 20 days of pregnancy on weekly body weight gain of pups.

Figure 8.　Effect of one week RF-EMR intrauterine exposure for on postnatal physiological development.

Figure 9.　Effect of tow week RF-EMR intrauterine exposure for on postnatal physiological development.

Figure 10.　Effect of three week RF-EMR intrauterine exposure for on postnatal physiological development.

3.8. Effect of RF-EMR 1800MHz GSM-like frequency on oxidant and antioxidant status in rats during different periods of pregnancy

In this study, it was investigated whether the 1800MHz GSM-like frequency exposure induced oxidative stress alters the antioxidant enzyme and hormone activity for scavenging free radicals. Antioxidant activities were determined biochemically and by using commercial ELISA kits. GD 0-GD 7 periods of intrauterine exposure to RF-EMR showed that the oxidant and antioxidant activity in all experimental groups remain consistent. After 2h/ day intrauterine exposure from GD 0-GD 14 GSH-PX activity was significantly lower than the observation in the control group. While 1h/day exposure showed no effect. MDA level was higher significantly in both exposure groups in comparison to control group. GD 0-GD 20 intervals showed a significant decrease in MT level after 2h/day intrauterine exposure and significant decrease in GSHPX activity in both exposure groups in comparison to control group. Both intrauterine exposure groups showed a significant increase in MDA level compare to control group. The results are shown in (Table 6).

4. Discussion

Fetal growth is one of the most sensitive stages of growth to electromagnetic radiation because developing tissues and organogenesis is more sensitive to harmful agents than those of adults. There are no consistent results, until now, about the effect of RF-EMR on prenatal and postnatal development due to a difference in experimental design, including SAR, frequencies, duration of exposure and short or long-term exposure although all the frequencies used within the microwave range. Our study differs from previous studies in that we investigated the biological effects of frequency 1 800 MHz GSM RF-EMR through different periods of pregnancy and its effect on the growth and development of embryos during pregnancy and after pregnancy. We elucidated that 1 800 MHz GSM radiation during pregnancy caused some detrimental effects on prenatal development as represented by significant unequal and asymmetrical distribution of implantation sites in both uterine horns of exposed groups except the control group, reduction in foetal weight with some anomalies as well as induction of oxidative stress in pregnant animals, decrease in weekly body weight gain and some negative effect on functional and physiological development. Previous studies have addressed various aspects of the impact of 1800 MHz frequency during pregnancy. For instance, Tomruk et al. found that exposing rabbits within middle stage of pregnancy for 15 min. /day for seven days to 1 800 MHz GSM-like signals lead to oxidative destruction in hepatic tissue as a result of production of free radicals in pregnant animal[19]. Another study performed by other Turkish team indicated that 1 800 MHz GSM-like signal’s exposure of non-pregnant and pregnant rabbits for seven days (15 min. /day) resulted in producing free radicals who induce lipid peroxidation and oxidative DNA damage [15], Foetal exposure to 800-1900 MHz-rated radiofrequency radiation from cellular phones lead to changes in behavioural and neurophysiological parameters (impaired memory, hyperactive and decreased anxiety) that persist into adulthood when mice exposed during pregnancy [9]. Our findings were similar with other recent studies on inutero exposure effect(s) of Wi-Fi frequency 2.45 GHz on pregnant animals and their infants (pre & postnatal developments). In one of such study, mice exposed to 2.45 GHz EMF for 2h/day for 30 days as (pre-mating period 22 day, mating period 5 days and postmating 3 days) exhibited asymmetrical and unequal implantation sites in uterine horns in exposed groups compared to control and induced DNA break in brain cells[20]. In another study mice exposed to 2.45GHz microwave in-utero irradiation continuous wave 2h/ day for 45 days at SAR 0.023023 W/kg as (pre-mating period 20 days, 5 days during mating and 20 days post-mating periods), this study showed that the MW irradiation induce oxidative stress which suppresses implantation (unequal/asymetrical distribution of embryos were observed in the uterine horns) and lead to embryonic deformity in case pregnancy continues and lead to DNA strand break in brain cells[21]. Also our findings is in agreement with [4] who found that pregnant rats exposed to 2450 MHz EMF resulted in postnatal growth restriction and delayed puberty in female rats due to induce oxidative stress in brain and ovarian tissues. Our results can be interpreted that the susceptibility for subfertility and prenatal growth impairement due to early embryonic death and resorption of embryo during second week of pregnancy was as a consequence of intrauterine irradiation inducing oxidative stress which play imprtant role in implantation and embryonic growth via inhibition of specific heat shock proteins (Hsp) such as Hsp70 and Hsp105. Hsp70 plays a critical role in fertilization and early embryonic development in mammalian. Furthermore, Hsp may also serve as a protective role in embryo development since inhibition of Hsp70 caused a reduction in blastocyst development that may be mediated by high rate of apoptosis[22], while temporary and spatial changes in Hsp105 expression in pregnat rat uterus play an important physiological role in regulating embryo implantaion[23]. The finding that RF-EMR influences prenatal development reflects the influence of many Hsps should be investigated further more. Oxidative stress is caused by the excessive production of free radicals or from unbalance of oxidant/antioxidant system. Our results data clearly show that RFEMR (1800 MHz) at low SAR leads to decrease in GSH-PX activity & MT levels as antioxidant enzyme and free radical scavenger as well as increase in MDA level, a biomarker for lipid peroxidation and tissue damage due to extensive production of peroxides and free radicals that cause toxic effect and damges all cell components for instance protein, lipids and DNA.

Reactive oxygen species (ROS) play an important role in the physiological reproductive functions such as maturation, ovarian steroidogenesis, corpus luteal function, fertilization, embryo development and pregnancy[24]. Despite the huge reports indicating, non-ionizing radiation induced oxidative stress and its effect on implantation, prenatal development and pregnancy, the precise mechanism of action of RF-EMR in inducing early embryo loss, malformation and impare pregnancy still unclear. More detailed studies should be conducted to determine the specific free radicals and Hsps contribution to prenatal development in different stages of embryogenesis. Postnatal development results showed a decrease in weekly body weight gain and delay in some functional and physiological development such as teeth & hair appearance, eyeopening and prolonged weaning period. This may be as a result of interference between the impact of oxidative stress and growth factors such as an insulin growth factors-I(IGF-I in the body. Our result is consistent with that of Dundar et al. which showed 50 Hz exposure resulted in growth restriction, delay puberty and reduced IGF-I level in female rats and probably associated with direct toxic effect of electric field on target organs[25]. IGF-I have a very important role in foetal and postnatal development. The variation in IGF-I levels associated with EMF exposure have been reported in few studies. Sangun et al. found that female rats exposed to 2.45 GHz during pregnancy, and postnatal development showed a decrease IGF-I level in the postnatal group compared to control animals as a result of induction of oxidative stress[4]. Picinato et al. found that MT via activation of MT 1 receptor regulates growth and differentiation of pancreatic islets by activating signaling pathway of IGF-I and insulin receptors[26]. In 2009, Oner conducted a study to investigate whether or not MT has a role in the prevention of carbontetrachlorid induced hepatotoxicity in rats by IGF expression. They found that MT secreted from pineal gland increases IGF-I was releasing and prevents hepatic damage[27]. Postnatal growth restriction in our experiment may be interpreted as due to decrease in MT level in experiment of three-week exposure to irradiation, which has the effect on releasing of IGF-I that have an important role in postnatal growth. Our results are in contrast with other recent studies. For instance, mice were intrauterine exposed 2h/day for 14 days, starting after five days of mating to Wi-Fi signal at 2.45 GHz, SAR 4W/kg and the result revealed no effect on pregnancy outcome due to prenatal exposure to Wi-Fi signals[28]. In another study, rats were in-uetro exposed to 2.45 GHz Wi-Fi signals (2h/day for 18 days at SAR 0.08, 0.4 and 4W/kg) to investigate the pre- and postnatal effect of Wi-Fi signals and no abnormalities were noted in pregnant rats, no signs of toxicity in the pre-and postnatal development of pups [13]. In a study conducted in France rats were exposed to 2.45 GHz Wi-Fi signals as whole-body exposure of free-moving rats for 1h/day for 3 weeks (male) two weeks (female) during sexual maturation, and the study suggested that Wi-Fi signals have no effect on male and female fertility at” WB SAR values up to the ICNIRP critical level”[29]. The current study, exposure to RF-EMR 1800 MHz during different periods of pregnancy resulted in different effects on pregnancy outcome and delayed in normal, functional and physiological postnatal development in pups from intrauterine irradiated dams. The frequency and SAR were used in our experiment were within safty limits of ICNIRP & IEEE guidelines. According to experiment results, we suggest that long-term exposure to EMF within microwave frequency during a pregnancy lead to chronic stress which in turn may cause some detrimental effects on pre- and postnatal development via different pathways, for that an additional studies need to be conducted to clarify the actual pathways that involve in such harmful effects.

Declare of interest statement

We declare that we have no conflict of interest.

Aknowledgement

The authors are indebted to our research assistance at a histopathology laboratory for their help. The project was fully supported by Faculty of Veterinary Medicine of Universiti Malaysia Kelantan.

References

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1800 MHz GSM frequency

Pre-& postnatal development

Rats

doi:Document heading 10.1016/j.apjr.2015.12.003

*Corresponding author:Ali S. H. Alchalabi, Faculty of Veterinary Medicine, UMK City Campus, Pengkalan Chepa, Locked Bag36, 16100 Kota Bharu, Kelantan, Malaysia.