|Year : 2014 | Volume
| Issue : 3 | Page : 185-191
Reduction in mortality and teratogenicity following simultaneous administration of folic acid and vitamin E with antiepileptic, antihypertensive and anti-allergic drugs
Shahana Wahid1, Rafeeq Alam Khan2, Zeeshan Feroz3
1 Department of Pharmacology, University of Karachi, Karachi 75270, Pakistan
2 Department of Basic Medical Sciences, College of Medicine, King Saud Bin Abdulaziz University for Health Sciences, Jeddah, KSA
3 Department of Basic Sciences, College of Science and Health Professions, King Saud Bin Abdulaziz University for Health Sciences, Riyadh, KSA
|Date of Submission||31-Mar-2013|
|Date of Decision||19-Aug-2013|
|Date of Acceptance||28-Sep-2013|
|Date of Web Publication||24-Jun-2014|
Rafeeq Alam Khan
Department of Basic Medical Sciences, College of Medicine, King Saud Bin Abdulaziz University for Health Sciences, Jeddah
Source of Support: Department of Pharmacology, Faculty of Pharmacy,
University of Karachi, Conflict of Interest: None
| Abstract|| |
Aim: The present study was designed to evaluate the teratogenic effects on breeding pattern in mice following administration of lamotrigine (LT) and levetiracetam (LV) and its combination with anti-hypertensive and anti-allergic drugs. Moreover, possibility of decrease in teratogenic effect was also evaluated upon simultaneous administration of these combinations with folic acid or vitamin E. Materials and Methods: The study was carried out on mature female mice housed in polycarbonate breeding cages. Drugs were administered continuously till the birth of neonates and pups were examined for any teratogenic potential of drugs. Results: The results of the study reveals that teratogenic effects of lamotrigine, methyldopa and loratadine (LTMLO) combination were seems to be reduced upon simultaneous administration with folic acid, while addition of vitamin E was found to be more effective in reducing the mortality rate of levetiracetam, methyldopa and loratadine combination. Conclusion: Teratogenic effects of LTMLO combination were better prevented by folic acid. However, further studies on large number of animals and humans are required before reaching to definite conclusion.
Keywords: Antiepileptic drugs, folic acid, teratogenicity, vitamin E
|How to cite this article:|
Wahid S, Khan RA, Feroz Z. Reduction in mortality and teratogenicity following simultaneous administration of folic acid and vitamin E with antiepileptic, antihypertensive and anti-allergic drugs. J Pharm Bioall Sci 2014;6:185-91
|How to cite this URL:|
Wahid S, Khan RA, Feroz Z. Reduction in mortality and teratogenicity following simultaneous administration of folic acid and vitamin E with antiepileptic, antihypertensive and anti-allergic drugs. J Pharm Bioall Sci [serial online] 2014 [cited 2020 Aug 10];6:185-91. Available from: http://www.jpbsonline.org/text.asp?2014/6/3/185/130955
Epilepsy is the leading neurological disorder in the world affecting approximately 45 million people world-wide.  Its prevalence in Pakistan is around 1%. , Numerous epidemiological studies have shown that the offspring's of epileptic mothers are at higher risk of congenital malformations as compared to general population; , therefore, the selection of drugs in pregnancy is limited due to several maternal physiologic changes affecting behavior. 
The most important adverse effect of the drug usage during pregnancy is the teratogenicity, hence knowledge of human embryology is essential to highlight the critical periods where major birth defects may arise, however late exposure to a drug during fetal development may not cause anatomical defects but may cause various functional abnormalities at biochemical and physiological level or behavioral defects. Many of these minor defects are not apparent at birth and may be dormant until later in life. ,
ICH (International Conference on Harmonization) has suggested three-segment strategy guideline, for the reproductive toxicity of drugs. Segment I is related to the recognition of effects on fertility, segment II is related to the detection of teratogenicity or embryo toxicity and segment III is related to peri- and post-natal study designed to explore developmental defects during late pregnancy and lactation. New born may face mainly four types of adverse effects after exposure to drugs during pregnancy i.e., death (embryo-fetal resorption, abortion, stillbirth or post-natal mortality), growth retardation (low birth weight or depressed post-natal growth), malformation and functional deficit. , Researchers have suggested several mechanisms for teratogenicity such as oxidative stress, neural crest cell disruption, folate antagonism, endocrine disruption, specific receptor-or enzyme-mediated teratogenesis, excessive apoptosis, genetic defects and interference with energy supply and osmolality. ,
Management of epilepsy during pregnancy is the main therapeutic challenge which must be balanced with the maternal and fetal risks associated with uncontrolled seizures so as to minimize the risk of teratogenicity. , None of the antiepileptic drugs (AEDs) are considered as to be safe and are categorized by Food and Drug Administration as class C or D drugs.  Furthermore, associated diseases complicate treatment plan of epilepsy during pregnancy, exposing fetus to increased risks of adverse effects.  Similarly, drugs in combination may cause diverse effects that are not recognized in individuals taking sole agent. 
Epileptic women with comorbid diseases take several drugs not only to control seizures, but for other disorders. Information regarding teratogenicity of individual AEDs is available; however, teratogenic potential of individual AEDs drugs with other commonly used drugs in pregnancy is not known. 
The present study was specifically designed to assess the teratogenic effects of lamotrigine (LT) and levetiracetam (LV) when used in combination with methyldopa and loratadine (LO) during pregnancy. LT and LV were selected since these were considered to be safest among AEDs.
Similarly methyldopa and LO were also considered to be safer and commonly prescribed in pregnancy for hypertension and allergic rhinitis, while folic acid and vitamin E was added to these combinations to reduce the risk of teratogenicity of these combinations.
Folic acid and vitamin E were selected on the basis of previous reports for reduction of teratogenic effect when co-administered. Folic acid produces several metabolites required to synthesize and repair deoxyribonucleic acid (DNA) and is necessary to express cell division which is intensified during pregnancy. Folic acid is particularly imperative in aiding rapid cell division and growth during infancy, pregnancy and lactation; hence, folic acid deficiency impairs DNA synthesis and cell division, affecting hematopoietic cells and neoplasm because of rapid cell division.  Folic acid supplementation shows to decrease the occurrence of neural tube defects (NTDs) or spina bifida, heart anomalies, urinary tract anomalies, omphalocele and anal atresia and some congenital anomalies. ,
Folic acid may be given to prevent congenital malformations including NTDs.  Vitamin E mainly acts as an antioxidant and is particularly vital for the protection of heart and circulation, nerves, muscles and red blood cells since neutralizes potentially damaging derivatives of free radicals formed via redox cycles. , Combination of vitamins C and E reduces the foetal malformation rate and tissue damage related to oxygen radical and improves foetal outcome. While in preeclampsia, plasma vitamin E can further prevent oxidative damage. 
| Materials and Methods|| |
The study was conducted in the Department of Pharmacology, Faculty of Pharmacy, after approval from Board of Advance Study and Research University of Karachi.
The study was carried out on mature female mice housed in polycarbonate breeding cages. Rodent chow and filtered tap water was provided ad libitum, whereas male mice were used only for matting until pregnancy and then caged separately near parturition. Animals were kept in a controlled climatic room (25 ± 2°C), during the entire study in an alternating 12-h light/dark cycle. The use of animals in this experiment was approved by Research Committee Department of Pharmacology in accordance with the guidelines of National Advisory Committee for Laboratory Animal Research  and National Institute of Health for use of laboratory animals. 
Experimental design and dosing
Animals were uniformly divided into 13 groups, each comprising of 7 female mice, about 3 to 4 female mice were replaced in some groups where pregnancy was not observed after 2 months. One group served as control while the other groups received the drug individually and in combination. Drugs were administered orally in normal therapeutic doses except folic acid and vitamin E until the birth of pups.
It is recommended that folic acid may be given to all women with childbearing age in a dose between 0.4 and 4 mg/day. It is suggested that 5 mg folic acid should be prescribed to women with epilepsy planning pregnancy.  Some neurologists usually prescribe 1 mg/day.  The recommended dietary allowance of vitamin E is 22-30 mg/day which is adequate to fulfil daily requirement of adult healthy individuals, pregnant and lactating women. 
However, in the present study folic acid and vitamin E were used in doses about two fold higher than recommended doses. LT was given in the dose of 6.66 mg/kg, LV 50 mg/kg, LO 0.33 mg/kg, methyldopa 16.66 mg/kg, folic acid 0.166 mg/kg and vitamin E 1 mg/kg. Water soluble drug LV was dispensed in distilled water while rests of the drugs were dispensed in 0.02 ml of 10% dimethyl sulfoxide (DMSO) below the oral permeable limit, whereas α-tocopherol was administered in the form of oily liquid.
Animals received drugs according to following pattern
- Group A: Control group received DMSO.
- Group B: LT.
- Group C: LV.
- Group D: Methyldopa (M).
- Group E: LO.
- Group F: Folic acid (F).
- Group G: Vitamin E (E).
- Group H: Lamotrigine-methyldopa-loratadine (LTMLO).
- Group I: Lamotrigine-methyldopa-loratadine-folic acid (LTMLOF).
- Group J: Lamotrigine-methyldopa-loratadine-vitamin E (LTMLOE).
- Group K: Levetiracetam-methyldopa-loratadine (LVMLO).
- Group L: Levetiracetam-methyldopa-loratadine-folic acid (LVMLOF).
- Group M: Levetiracetam-methyldopa-loratadine-vitamin E (LVMLOE).
Controls animals were treated with the equivalent amount of vehicle according to body weight used to deliver the respective drugs. Each pair was housed to permit matting and the pairing day was counted as day 1 for the administration of drugs to female mice only.
The drugs were administered continuously until the birth of neonates. Pups were examined for teratogenic potential of drugs after birth. For non-perceived mice, two mice were sacrificed from each group and dissection of uterus was done to proceeded histological studies.
Evaluation of teratogenicity
Apparent health of animals and pups after birth was monitored at an interval of 1 week under the laboratory environment until 21 st day of birth (the day at which usually pups are able to feed). Other parameters observed were maternal weight, number of live and dead fetuses, their mortality rate, fetal weight, gross fetal morphologic malformations, visceral anomalies and skin allergic reactions, which were compared with the control group.
Microscopic uterus tissue examination
Two female mice from each group were made insensible by spinal cord injury. Dissection of uterus was carried out and specimen of uterus without ovary was preserved in 10% buffered formalin for further processing. Suitable blocks of these organs were taken, fixed, processed and the sections were cut for microscopic examination of uterus after administration of drugs for a maximum period of 90 days.
Preparation of slide
Representative blocks from uteri were cut from each sample. Tissue sections of 3-4 μ thickness were cut from the wax blocks by rotary manual microtome. The tissue sections were mounted on slides and were dried gently by pressing with filter paper. The mounted slides were placed initially in an incubator at 37°C overnight to dry. This was followed by standard staining procedure for histopathological analysis.
Data was statistically analyzed using one sample t-test and results were considered as significant when P < 0.05.
| Results|| |
Evaluation of teratogenicity
Teratogenic potential was evaluated as per parameter listed in [Table 1].
|Table 1: Teratogenic effects of individuals drugs, lamotrigine, levetiracetam and their combinations|
Click here to view
Change in apparent health
No apparent change in health of animals pups received individual or combination drugs was observed [Figure 1]. However, pups of 1 out of 10 females received DMSO and LTMLO showed skin allergy [Figure 2].
None of the group revealed any facial defects except groups of LT (10.94%) and LTMLO (10.71%) where minor facial defects were observed. Incomplete formation of hind and fore limb and mouth was also observed in death fetuses of the two groups [Figure 3]a and b.
|Figure 3: (a) Lamotrigine group fetus; (b) Lamotrigine-methyldopa-loratadine group fetus|
Click here to view
Slow growth of new born
Slow growth was observed in terms of time taken to fully grow to adult mice, full growth was usually observed within 2 months of birth in normal animals. Groups of LTMLO, LTMLOF LVMLO and LVMLOE revealed slow growth and even simultaneous administration of folic acid to animals received LT failed to improve slow growth. However, vitamin E produced a prominent growth enhancement effect on the live fetuses of animals received LT in combination. However, animals received LV in combination did not reveal significant reduction in slow growth by simultaneous use of vitamin E, but folic acid produced a prominent effect on growth enhancement of the live fetuses. Animals received individual drugs did not revealed slow growth.
Fetal weights and still births
[Table 1] shows average weight in gram ± standard error of the mean. Average weight of live fetus was not significantly decreased in any animal groups as compared to control. The use of folic acid and vitamin E also did not produce any significant effect on weight. Weight of all dead fetuses was significantly low in LTMLO, LTMLOE, LVMLO, LVMLOF and LVMLOE groups when compared to dead fetuses' weight in control animals.
Pups of animal groups received DMSO, LT and LV revealed 5.36%, 17.07% and 17.07% mortality rate respectively. Mortality rate in animals received LT in combination with methyldopa and LO was increased to 35.71%; however, mortality rate was reduced to 0% when folic acid was added to this combination, whereas addition of vitamin E to this combination also reduced mortality rate significantly by 17.69% [Figure 4].
Mortality rate was increased to 29.69% in animals received LV in combination with methyldopa and LO; furthermore there was no significant change in mortality rate after the addition of folic acid to the combination. However, addition of vitamin E to LV with methyldopa and LO significantly reduced mortality to 14.29%.
Microscopic tissue examination
Gross examination of mice uterus did not reveal any macroscopic changes in any group except LTMLOF where an abnormal massive body was found in a mouse. In LVMLO, LVMLOF and LVMLOE groups, one mouse in each group have embryonic tissue showing pregnancy.
[Table 2] reveals the description of microscopic changes in uterus of various animal groups in the study. Microscopic examination of the uterus of control animals did not reveal any changes [Figure 5]. Similarly no changes were observed in animals received methyldopa, folic acid, LO and vitamin E; however, animals treated with LT showed hemorrhage in stroma and slightly enlarged lumen [Figure 6]. Microscopic examination of uterus in animals received LTMLOF combination revealed fibrous fatty tissue densely infiltrated by inflammatory cells with areas of necrosis [Figure 7]. Animals received LVMLO, LVMLOF and LVMLOE combinations showed normal embryonic tissues showing pregnancy.
|Figure 5: Uterus showing no microscopic changes (a) Lumen lined with tall columnar epithelial cells; (b) Endometrium; (c) Endometrial glands lined by cuboidal epithelium glands may display congromeleration; (d) Myometrium|
Click here to view
| Discussion|| |
Management of epilepsy in pregnant women is quite complicated due to the risk for unfavorable outcomes on pregnancy and newborn, which is further intensified if the mother has other co-existence diseases. Children of women with epilepsy has overall incidence of congenital malformations that is about threefold more than normal healthy women. 
Data concerning teratogenicity of AEDs used in combination with other therapeutic class of drug is rare due to which present study was focused on the evaluation of teratogenicity by AEDs when used in combination with drugs commonly used in pregnancy for hypertension and allergic rhinitis.
The overall health of new born animals was normal except few groups where still births or functional deficit were found. Animals of two groups received DMSO and LTMLO revealed skin allergy in about 20% of animals and even the presence of LO failed to inhibit allergy. Evidence of skin allergy had been reported following administration of LT alone which regressed after the concomitant administration of methyl prednisolone. , However, no skin allergy was found in the fetus of the other groups.
The animal groups received methyldopa, LO, folic acid and vitamin E alone did not reveal any malformation, growth retardation and still birth; however, animals received LT revealed malformations, growth retardation and still births which was in accordance to previous studies. ,,, LT when given in combination with methyldopa and LO not only revealed malformation, but also showed still births and significant reduction in birth weights of still fetuses when compared to control. Moreover, growth rate of fetuses in the animals of this group was also slow. However, when folic acid was added to the above combination, malformations and still births were significantly reduced but growth rate was unaffected. Although addition of vitamin E to LTMLO combination, inhibited malformations, improved growth rate and reduced still births.
Animals received LV revealed growth retardation and still births which was in accordance to previous studies. ,, Animals received LV in combination with methyldopa and LO revealed still births, significant reduction in weights of still births and slow growth rate of fetuses when compared with control. However, when folic acid was added to the above combination, growth rate was improved but rate of still births remained unaffected, while there was insignificant improvement in weight of still fetuses. Addition of vitamin E to LVMLO combination reduced still births significantly while growth rate remain unaffected.
Results of the present study showed that folic acid has been found effective in preventing still births and growth retardation, which was in accordance to previous studies showing protective efficacy in reducing various anomalies. ,, Similarly, vitamin E also reduced teratogenic effects in present study, which were in accordance to the previous studies. ,,,
Microscopic examination of the uterus of animals received LT, LO individually and LTMLOF in combination revealed remarkable changes highlighting that this combination trigger cellular changes such as necrosis; however, further studies are required to elaborate the cellular effects.
| Conclusion|| |
Results of present study reveals that poly pharmacy during pregnancy may be related with increased teratogenic potential of AEDs; however, teratogenic effects can be reduced by both folic acid and vitamin E since both have been found effective in reducing various teratogenic effects. Teratogenic effects induced in LTMLO combination were more prevented by folic acid. It is suggested that women on AEDs expecting pregnancy may take folic acid 10 mg and vitamin E 60 mg daily to avoid teratogenic effects of these drugs. In addition, prompt reporting system should be established as part of program to guide safe use of these drugs during pregnancy; however, further studies on large number of animals are required to reach at definite conclusion.
| Acknowledgment|| |
We would like to thank Dr. Muhammad Arif and Dr. Tazeen from Baqai Medical College and Dow University of Health Sciences for their technical assistance in the dissection procedure and histopathological studies.
| References|| |
|1.||French JA, Pedley TA. Clinical practice. Initial management of epilepsy. N Engl J Med 2008;359:166-76. |
|2.||Aziz H, Ali SM, Frances P, Khan MI, Hasan KZ. Epilepsy in Pakistan: A population-based epidemiologic study. Epilepsia 1994;35:950-8. |
|3.||Aziz H, Güvener A, Akhtar SW, Hasan KZ. Comparative epidemiology of epilepsy in Pakistan and Turkey: Population-based studies using identical protocols. Epilepsia 1997;38:716-22. |
|4.||Finnell RH, Dansky LV. Parental epilepsy, anticonvulsant drugs, and reproductive outcome: Epidemiologic and experimental findings spanning three decades; 1: Animal studies. Reprod Toxicol 1991;5:281-99. |
|5.||Arpino C, Brescianini S, Robert E, Castilla EE, Cocchi G, Cornel MC, et al. Teratogenic effects of antiepileptic drugs: Use of an International Database on Malformations and Drug Exposure (MADRE). Epilepsia 2000;41:1436-43. |
|6.||Ward RM. Pharmacology of the maternal-placental-fetal-unit and fetal therapy. Prog Pediatr Cardiol 1996;5:79-89. |
|7.||Finnell RH. Teratology: General considerations and principles. J Allergy Clin Immunol 1999;103:S337-42. |
|8.||Rakusan K. Drugs in pregnancy: Implications for a cardiologist. Exp Clin Cardiol 2010;15:e100-3. |
|9.||van Gelder MM, van Rooij IA, Miller RK, Zielhuis GA, de Jong-van den Berg LT, Roeleveld N. Teratogenic mechanisms of medical drugs. Hum Reprod Update 2010;16:378-94. |
|10.||Lindhout D. Pharmacogenetics and drug interactions: Role in antiepileptic-drug-induced teratogenesis. Neurology 1992;42:43-7. |
|11.||Morrell MJ. Epilepsy in women. Am Fam Physician 2002;66:1489-94. |
|12.||Luef G. Female issues in epilepsy: A critical review. Epilepsy Behav 2009;15:78-82. |
|13.||Bruno MK, Harden CL. Epilepsy in pregnant women. Curr Treat Options Neurol 2002;4:31-40. |
|14.||Jacqz-Aigrain E, Koren G. Effects of drugs on the fetus. Semin Fetal Neonatal Med 2005;10:139-47. |
|15.||Menon SJ. Psychotropic medication during pregnancy and lactation. Arch Gynecol Obstet 2008;277:1-13. |
|16.||Pack AM, Davis AR, Kritzer J, Yoon A, Camus A. Antiepileptic drugs: Are women aware of interactions with oral contraceptives and potential teratogenicity? Epilepsy Behav 2009;14:640-4. |
|17.||Kamen B. Folate and antifolate pharmacology. Semin Oncol 1997;24 5 Suppl 18:S18-30-S18-39. |
|18.||Detrait ER, George TM, Etchevers HC, Gilbert JR, Vekemans M, Speer MC. Human neural tube defects: Developmental biology, epidemiology, and genetics. Neurotoxicol Teratol 2005;27:515-24. |
|19.||Meijer WM, de Walle HE, Kerstjens-Frederikse WS, de Jong-van den Berg LT. Folic acid sensitive birth defects in association with intrauterine exposure to folic acid antagonists. Reprod Toxicol 2005;20:203-7. |
|20.||Lakshmi S, Sunanda K. Effect of anti-epileptic drugs in pregnancy and teratogenesis. Indian J Clin Biochem 2008;23:267-71. |
|21.||Liebler DC. The role of metabolism in the antioxidant function of vitamin E. Crit Rev Toxicol 1993;23:147-69. |
|22.||van Staden AM, van Rensburg CE, Anderson R. Vitamin E protects mononuclear leucocyte DNA against damage mediated by phagocyte-derived oxidants. Mutat Res 1993;288:257-62. |
|23.||Min J, Park H, Park B, Kim YJ, Park J, Lee H, et al. Paraoxonase gene polymorphism and vitamin levels during pregnancy: Relationship with maternal oxidative stress and neonatal birthweights. Reprod Toxicol 2006;22:418-24. |
|24.||National Advisory Committee for Laboratory Animal Research. Guidelines on the Care and Use of Animals for Scientific Purposes; 2004. p. 24. |
|25.||National Research Council. Guide for the Care and Use of Laboratory Animals. Washington, DC: National Academy Press; 1996. p. 1-7. |
|26.||Tettenborn B. Management of epilepsy in women of childbearing age: Practical recommendations. CNS Drugs 2006;20:373-87. |
|27.||Schachter SC. Currently available antiepileptic drugs. Neurotherapeutics 2007;4:4-11. |
|28.||Boskovic R, Gargaun L, Oren D, Djulus J, Koren G. Pregnancy outcome following high doses of Vitamin E supplementation. Reprod Toxicol 2005;20:85-8. |
|29.||Meador K, Reynolds MW, Crean S, Fahrbach K, Probst C. Pregnancy outcomes in women with epilepsy: A systematic review and meta-analysis of published pregnancy registries and cohorts. Epilepsy Res 2008;81:1-13. |
|30.||Dooley J, Camfield P, Gordon K, Camfield C, Wirrell Z, Smith E. Lamotrigine-induced rash in children. Neurology 1996;46:240-2. |
|31.||Gücüyener K, Türktaº I, Serdaroglu A, Ezgü FS. Suspected allergy to lamotrigine. Allergy 1999;54:767-8. |
|32.||Dolk H, Jentink J, Loane M, Morris J, de Jong-van den Berg LT, EUROCAT Antiepileptic Drug Working Group. Does lamotrigine use in pregnancy increase orofacial cleft risk relative to other malformations? Neurology 2008;71:714-22. |
|33.||Tomson T, Battino D. Teratogenic effects of antiepileptic drugs. Seizure 2008;17:166-71. |
|34.||Danielsson I, Lister L. A pilot study of the teratogenicity of vagus nerve stimulation in a rabbit model. Brain Stimul 2009;2:41-9. |
|35.||Jarita D, Errol F, Thomas M, Chanda K. Risk of malformation with combination of lamotrigine and low dose clonazepam for juvenile myoclonic epilepsy in pregnancy: A case report. Neurol Asia 2010;15:79-81. |
|36.||Czeizel AE, Bartfai Z, Banhidy F. Primary prevention of neural tube defects and some other congenital abnormalities by folic acid and multivitamins: History, missed opportunity and tasks. Ther Adv Drug Saf 2011;2:173-88. |
|37.||Cachia O, Léger CL, Boulot P, Vernet MH, Michel F, Crastes de Paulet A, et al. Red blood cell vitamin E concentrations in fetuses are related to but lower than those in mothers during gestation. A possible association with maternal lipoprotein (a) plasma levels. Am J Obstet Gynecol 1995;173:42-51. |
|38.||Scholl TO, Chen X, Sims M, Stein TP. Vitamin E: Maternal concentrations are associated with fetal growth. Am J Clin Nutr 2006;84:1442-8. |
|39.||Chen CS, Wells PG. Enhanced tumorigenesis in p53 knockout mice exposed in utero to high-dose vitamin E. Carcinogenesis 2006;27:1358-68. |
|40.||Wells PG, McCallum GP, Chen CS, Henderson JT, Lee CJ, Perstin J, et al. Oxidative stress in developmental origins of disease: Teratogenesis, neurodevelopmental deficits, and cancer. Toxicol Sci 2009;108:4-18. |
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
[Table 1], [Table 2]