Teratogenic effects of silver nanoparticles: gross anomalies.
The present study was undertaken to determine the dysmorphogenesis effects of AgNPs on fetal development after maternal exposure on gestational days (GD) 4-15 in mice.
MATERIAL& METHODS: Male and female Swiss albino mice with an average weight of 20-35gms and average age of 45days from different breeding colony were chosen randomly for conduction of this study from animal house, Department of Anatomy, Institute of Medical Sciences, Banaras Hindu University. Animals were housed individually in plastic cages with steel toppings (1:1 male female ratio) with early maintenance of 12hrs day and night cycle in the air conditioned animal house, the temperature was maintained around 20-250c with a minimum range of relative humidity of 55 [+ or -] 5%. Mice were feed on Hindustan liver diet pellets and tap water libitum with appropriate bedding made up of dry husk inside the plastic cage. Vaginal plugged female dams were considered as day "zero" of pregnancy. The pregnant female mice were weighted from day 0 to day 18 of gestational age and were kept individually in separate cages till day 18th of G.A for exteriorization by deep ether/chloroform anesthesia and collection of fetuses. Silver nanoparticle colloidal solution prepared by using (magnetic stirring and cooling method). (15) The mothers were administered with silver nanoparticle colloidal solution with adding 1.5 M NaCl solution as vehicle, 1 drop of 0.33% poly vinyl pyrollidone solution as a deaggregator agent and 0.002M Sodium Borohydride solution as a stabilizer agent, by oral gavages. The study was carried out in strict conformity with laws and regulation for animal experiment after getting approval from central animal ethical committee of the institute (No. Dean/ 2014/ CAEC/ 6). All Animals were treated with utmost humane care.
Characterization of silver nanoparticles.
Freshly prepared bigger size AgNPs colloidal solution was characterized by Dynamic Light Scattering. (16)
Dissection schedule, body malformation observation, photography and percentage evaluation All dams were subjected to exteriorization on GD 18. The fetuses were evaluated for signs of body malformation effects under dissecting microscope. 6 dams were taken to conduct experiment from each group. Each dam delivered almost 7 to 13 fetuses and all the fetuses were evaluated. Optical zoom 18.1 Mega pixel CMOS 3.2 vary-angle camera was used for photography. At the end percentage evaluated from each group with overall percentages compared between the groups. Overall percentage of live fetuses, overall percentage of dead fetuses and overall mean weight of live fetuses were calculated from each group.
RESULT: OBSERVATION: All foetuses exposed to 0.5, 1, 5, 10, 15 & 20mg/kg/day AgNPs group were evaluated for signs of body malformation and were compared with all foetuses of anionic double distilled water treated sham control group. Out of 65 foetuses from 0.5mg/kg/day AgNPs treated group 3 foetuses were observed resorption, 5 were observed IUGR. Out of 65foetuses from 1mg/kg/day AgNPs treated group 6 foetuses were observed resorption, 7 were observed I.U.G.R., 1undeveloped foetus with full product of conception including amniotic membrane and placenta observed severe haemorrhage within the body. (Fig. 3 a & b) Out of 57 foetuses from 5mg/kg/day AgNPs treated group 4 foetuses were observed limb malformation, proboscis with phocomelia. (Fig.3c,d&e), 3 were observed closed type neural tube defect (Fig. 3h), 10 were observed resorption and 10 were observed IUGR, Out 57 foetuses from 10mg/kg/day AgNPs treated group 2 foetuses were observed Amelia (Fig. 3f), 2 were observed foot and tail vein haemorrhage (Fig. 3i), 2were observed simple tail vein haemorrhage (Fig. 3j), 13 were observed resorption and 17 were observed I.U.G.R. Out of 57 foetuses from 15mg/kg/day AgNPs treated group 5 foetuses were observed limb anomaly like syndactyly, oligodactyly with extended limb anomaly (Fig. 3g), 13 foetuses were observed severe IUGR and external haemorrhages. (Fig. 3k), 24 foetuses were observed resorption, 26 foetuses were observed IUGR. Out of 61foetuses from 20mg/kg/day AgNPs treated group 2 foetuses were observed Omphalocele anomaly (Fig. 3l), 6 were observed Bidiscoidal placental anomaly. (Fig. 3m). 8 were observed resorption and 38 were observed I.U.G.R. Common sign and symptoms of body malformations of foetuses like resorption, IUGR and haemorrhagic consequences were seen in all treated dams foetuses consistently but the intensity of such was found increased as the dose increases.
Summary of calculated percentages of foetuses individual body malformations from control and treated groups.
Table 1: Summarizes total number of dams, total number of fetuses, type of fetus body malformations and their percentages from different groups.
Individual calculated percentages of fetal body malformations summarize in Table 2 showed that the intensity of such malformations increased with increase in dose of silver nanoparticles through repeated maternal oral gavages exposure whereas the percentage of normal decrease.
Table 2: Summarizes dose, total number of mothers, total number of foetuses, number of absorption, number of live foetuses.
Table 3: Summarizes the numbers of malformations from different groups.
F.N.-*IUGR-Intra Uterine Growth Retardation/*L. M.-Limb malformation/*CNTD-Close type neural tube defect/*FTVH-Foot and tail vein haemorrhage/*TVH- Tail vein haemorrhage/ *S. D. O. D. E. L. A. Syndactyly, oligodactyly, extended limb anomaly/*S. E. H. W. B-Severe external haemorrhages within body/*Ompha.-Omphalocele/*B. D. P. A.-Bidiscoidal placental anomaly.
Number of live fetuses and number of dead fetuses of control and treated group from table 2 analysis shows the number of live fetuses' percentages decreases from control group to 20mg/kg/day treated group. Whereas the number of dead fetuses percentages increases from control group towards 20mg/kg/day group with an exceptional case of increase of percentages of dead fetuses seen in 15mg/kg/day group.
Mean percentages of various malformations.
Graph 1: Showing mean and individual of percentages of various malformations from control and treated group fetuses.
Analysis of figure 2 shows the mean percentages of various malformations progressively increases from control towards 20 mg/kg/day group with an exceptional increase in 15mg/kg/day AgNPs treated group.
Overall percentages of IUGR.
Graph 2: Showing percentages of IUGR from control and treated groups.
The figure 3 analysis shows percentages of IUGR progressively increases from control toward 20mg/kg/day treated group.
Table 4: Summarizes dose, meant s.d. of crown rump length in fetuses and meant s.d. of weight of live fetuses in gms.
Graph 3: Showing meant s.d. of crown rump length of fetuses from control and treated groups.
Table 3 data Figure 4 and 5 analyses shows the mean[+ or -]s.d. of crown rump length and weight of live fetuses decreases progressively from control towards 20mg/kg/day group and with increase in dose.
Graph 4: Showing mean[+ or -]s.d. of weight of live fetuses from control and treated groups.
The figure 6 analysis shows percentages of resorption progressively increases from control toward 20mg/kg/day treated group.
Graph. 5: Showing percentages of Resorption from control and treated groups.
DISCUSSION: Silver nanoparticle ingestion causes detrimental effect on embryo fetus development. It also causes various malformations in fetuses when they grow in womb. The deleterious effects visualizes when they born after maternal exposure of AgNPs in prenatal stage. The various malformations fetus met with are limb malformations, snot mal development, virulent hemorrhages within the body, mid gut loop herniation like omphalocele, bidiscoidal placental anomaly, close type neural tube defect, resorption, intra uterine growth retardation, syndactyly, oligodactyly, foot and tail vein hemorrhages. These malformations hamper fetal weight, nutrition and crown rump length of fetuses. Various animal tests are done till today for sorting out the cause of malformations in fetus after silver nanoparticle ingestion through various routes. Maternal repeated oral exposure of AgNPs in pregnant Swiss albino micedams also causes various malformations in fetuses which are for the first time reported in our experiment. The oral ingestion tests of silver nanoparticles on maternal body cause's arousal of various malformations in exteriorized fetuses which increases in percentages with increase in dose. The data of present study says when 0.5, 1, 5, 10, 15 & 20 mg/kg/day silver nanoparticle colloidal solution is injected to pregnant Swiss albino mice it causes arousal of culminative toxicity in fetuses with various malformations. The percentages of such malformations increase with increase in dose of silver nanoparticles. Resorption is a common form of gross anomaly which increases with increase in dose of AgNPs and the increased percentages was evaluated in our study with the increase in dose though control group showed minimal percentage of resorption (3.70%). (Table 1 & 2; Fig. 1).
Intra uterine growth retardation also a common form of gross anomaly which visualized in fetuses while conducting the present study. The calculated percentages are seen progressively increasing with increase in dose of AgNPs as a consequence of this mean[+ or -]s.d. of fetal crown rump length and weight of live fetuses also increased progressively with increase in dose where control group also showed minimal percentages of IUGR (5.55%). (Table 1, 2 and Fig.2). Rest of the gross malformations like limb malformations, snot mal development, proboscis, phocomelia, virulent hemorrhages within the body, mid gut loop herniation like omphalocele, bidiscoidal placental anomaly, close type neural tube defect, syndactyly, oligodactyly, foot and tail vein hemorrhages and simple tail vein hemorrhages also seen increases in terms of percentages with increase in dose of silver nanoparticles. (Table 1& 2; Fig. 3) An exceptional growth of percentages of malformations like severe IUGR and hemorrhages within the body was met in the fetuses of 15mg/kg/day treated group during the course of experiment. (Table 1& 2; Fig. 3 with data underlined and bar diagram with arrow marked). Number and percentages of live fetuses seen decreasing with increase in dose of AgNPs. (Table 2).
Though there is hardly any data available regarding assessment of gross teratogenic anomalies of fetuses by silver nanoparticle colloidal solution after repeated maternal exposure testing our study confirms a strong evidence that silver nanoparticles certainly shows deleterious effects in fetuses after maternal oral exposure. For researcher benefit and public awareness over ignorance the potential adverse effects of nanosilver administration has been reported that, inovoof 50 particles per million hydrocolloids of nanoparticles of silver did not influence mortality, growth and development within 48 hrin 20 days old chicken embryos, C1718) but present study "AgNPs effects on fetus gross anomalies" tries to convince 0.5, 1, 5, 10, 15 & 20 mg/kg/day bigger silver nanoparticles of 20 to 100 nm size range in silver nanoparticle colloidal solution repeated oral administration and testing definitely have deleterious effects on embryo-fetus development which cause gross anomalies in fetuses of Swiss albino dams. It confirms repeated oral gavages silver nanoparticle colloidal solution testing on animals in pregnancy cause hampers of fetus natural growth in womb and fetus body morphology is insulted by this, and resulted in abnormal development.
CONCLUSION: The present study highly suggests the deleterious effects of nanosilver on embryo fetus development and thus a teratogen which is dose dependent.
ACKNOWLEDGEMENT: The author sincerely acknowledges University Grant Commission.
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Jyoti Prakash Pani , Rajniti Prasad , Deepika Joshi , Royana Singh 
[1.] Jyoti Prakash Pani
[2.] Rajniti Prasad
[3.] Deepika Joshi
[4.] Royana Singh
PARTICULARS OF CONTRIBUTORS:
[1.] Research Scholar, Department of Anatomy, IMS, BHU Varanasi, Uttar Pradesh.
[2.] Professor, Department of Paediatrics, IMS, BHU, Varanasi, Uttar Pradesh.
[3.] Professor & HOD, Department of Neurology, IMS, BHU, Varanasi, Uttar Pradesh.
[4.] Professor, Department of Anatomy, IMS, BHU, Varanasi, Uttar Pradesh.
FINANCIAL OR OTHER COMPETING INTERESTS: None
NAME ADDRESS EMAIL ID OF THE CORRESPONDING AUTHOR:
Dr. Royana Singh, Department of Anatomy, Institute of Medical Sciences, Banaras Hindu University, Varanasi-221005, Uttar Pradesh.
E-mail: firstname.lastname@example.org email@example.com
Date of Submission: 13/07/2015.
Date of Peer Review: 14/07/2015.
Date of Acceptance: 28/07/2015.
Date of Publishing: 31/07/2015.
Table 1 Groups Type of fetus body malformations Percentages (Total no. of fetuses) Control group a) Resorption (2) 3.70% b) I.U.G.R. (3) 5.55% 0.5mg/kg/day a) Resorption (3) 4.61% AgNPs Tr. Gr. b) I.U.G.R. (5) 7.69% 1mg/kg/day a) Resorption (6) 9.23% AgNPs Tr. Gr. b) I.U.G.R. (7) 10.76% c) Severe hemorrhage within body (1) 1.53% 5mg/kg/day a) Limb malformation& proboscis with 7.01% AgNPs Tr. Gr. IUGR (4) 5.26% b) Closed type NTD with IUGR (3) c) Resorption (10) 17.54% d) IUGR (10) 17.54% 10mg/kg/day a) a)Amelia with I.U.G.R. (2) 3.50% AgNPs Tr. Gr. b) b)Foot and tail vein hemorrhages 3.50% with I.U.G.R. (2) c) c)Simple tail vein hemorrhages 3.50% with I.U.G.R. (2) d) Resorption (13) 22.80% e) I.U.G.R. (17) 29.82% 15mg/kg/day a) Severe IUGR with external 22.80% AgNPs Tr. Gr. hemorrhages (13) 8.77% b) Syndactyly, oligodactyly, extended 42.10% limb anomalies with IUGR (5) c) Resorption (24) 45.61% d) I.U.G.R. (26) 20mg/kg/day a) Omphalocele with IUGR (2) 3.27% AgNPs Tr. Gr. b) Bidiscoidal placental anomalies 9.83% with IUGR(6) c) Resorption (18) 29.50% d) I.U.G.R. (38) 62.29% Table 2: Summarizes dose, total number of mothers, total number of foetuses, number of absorption, number of live foetuses. Table 2 Sl. Dose No. of Total no. No. of live No. mother of fetuses fetuses 1 Control 6 54 52 (96.29%) 2 0.5mg tr.gr. 6 65 62 (95.38%) 3 1mg tr.gr. 6 65 59 (90.76%) 4 5mg tr.gr. 6 57 47 (82.45%) 5 10mg tr.gr. 6 57 44 (77.19%) 6 15mg tr.gr. 6 57 33 (57.89%) 7 20mg tr.gr. 6 61 43 (70.49%) Table 3: Summarizes the numbers of malformations from different groups. Table 3 Malformations Resorption IU Severe L.M. CN Amelia GR Haemo. with TD Within phoco body melia 2 3 -- -- -- -- 3 5 -- -- -- -- 6 7 1 -- -- -- 10 10 -- 4 3 -- 13 17 -- -- -- 2 24 26 -- -- -- -- 18 38 -- -- -- -- Malformations Resorption FT TVH S.D. S.E.H. Omp B.D. VH O.D. W.B ha. P.A. E.A 2 -- -- -- -- -- -- 3 -- -- -- -- -- -- 6 -- -- -- -- -- -- 10 -- -- -- -- -- -- 13 2 2 -- -- -- -- 24 -- -- 5 13 -- -- 18 -- -- -- -- 2 6 Table 4 Sl. Dose Mean [+ or -] S.D. Mean [+ or -] No. of CRL (Crown S.D. of weight of rump length in live fetuses in mm.) gm. 1 Control 270 [+ or -] 15.15 1.253 [+ or -] 0.002 2 0.5mg/kg/day 265 [+ or -] 14.12 1.203 [+ or -] 0.002 3 1 mg/kg/day 260 [+ or -] 13.02 1.153 [+ or -] 0.002 4 5 mg/kg/day 230 [+ or -] 9. 02 0.792 [+ or -] 0.001 5 10 mg/kg/day 190 [+ or -] 7.22 0.784 [+ or -] 0.001 6 15 mg/kg/day 150 [+ or -] 5.02 0.732 [+ or -] 0.001 7 20 mg/kg/day 110 [+ or -] 3.22 0.701 [+ or -] 0.001
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|Title Annotation:||ORIGINAL ARTICLE|
|Author:||Pani, Jyoti Prakash; Prasad, Rajniti; Joshi, Deepika; Singh, Royana|
|Publication:||Journal of Evolution of Medical and Dental Sciences|
|Date:||Aug 3, 2015|
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