Journal of Clinical and Diagnostic Research, ISSN - 0973 - 709X

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Dr Bhanu K Bhakhri

"The Journal of Clinical and Diagnostic Research (JCDR) has been in operation since almost a decade. It has contributed a huge number of peer reviewed articles, across a spectrum of medical disciplines, to the medical literature.
Its wide based indexing and open access publications attracts many authors as well as readers
For authors, the manuscripts can be uploaded online through an easily navigable portal, on other hand, reviewers appreciate the systematic handling of all manuscripts. The way JCDR has emerged as an effective medium for publishing wide array of observations in Indian context, I wish the editorial team success in their endeavour"



Dr Bhanu K Bhakhri
Faculty, Pediatric Medicine
Super Speciality Paediatric Hospital and Post Graduate Teaching Institute, Noida
On Sep 2018




Dr Mohan Z Mani

"Thank you very much for having published my article in record time.I would like to compliment you and your entire staff for your promptness, courtesy, and willingness to be customer friendly, which is quite unusual.I was given your reference by a colleague in pathology,and was able to directly phone your editorial office for clarifications.I would particularly like to thank the publication managers and the Assistant Editor who were following up my article. I would also like to thank you for adjusting the money I paid initially into payment for my modified article,and refunding the balance.
I wish all success to your journal and look forward to sending you any suitable similar article in future"



Dr Mohan Z Mani,
Professor & Head,
Department of Dematolgy,
Believers Church Medical College,
Thiruvalla, Kerala
On Sep 2018




Prof. Somashekhar Nimbalkar

"Over the last few years, we have published our research regularly in Journal of Clinical and Diagnostic Research. Having published in more than 20 high impact journals over the last five years including several high impact ones and reviewing articles for even more journals across my fields of interest, we value our published work in JCDR for their high standards in publishing scientific articles. The ease of submission, the rapid reviews in under a month, the high quality of their reviewers and keen attention to the final process of proofs and publication, ensure that there are no mistakes in the final article. We have been asked clarifications on several occasions and have been happy to provide them and it exemplifies the commitment to quality of the team at JCDR."



Prof. Somashekhar Nimbalkar
Head, Department of Pediatrics, Pramukhswami Medical College, Karamsad
Chairman, Research Group, Charutar Arogya Mandal, Karamsad
National Joint Coordinator - Advanced IAP NNF NRP Program
Ex-Member, Governing Body, National Neonatology Forum, New Delhi
Ex-President - National Neonatology Forum Gujarat State Chapter
Department of Pediatrics, Pramukhswami Medical College, Karamsad, Anand, Gujarat.
On Sep 2018




Dr. Kalyani R

"Journal of Clinical and Diagnostic Research is at present a well-known Indian originated scientific journal which started with a humble beginning. I have been associated with this journal since many years. I appreciate the Editor, Dr. Hemant Jain, for his constant effort in bringing up this journal to the present status right from the scratch. The journal is multidisciplinary. It encourages in publishing the scientific articles from postgraduates and also the beginners who start their career. At the same time the journal also caters for the high quality articles from specialty and super-specialty researchers. Hence it provides a platform for the scientist and researchers to publish. The other aspect of it is, the readers get the information regarding the most recent developments in science which can be used for teaching, research, treating patients and to some extent take preventive measures against certain diseases. The journal is contributing immensely to the society at national and international level."



Dr Kalyani R
Professor and Head
Department of Pathology
Sri Devaraj Urs Medical College
Sri Devaraj Urs Academy of Higher Education and Research , Kolar, Karnataka
On Sep 2018




Dr. Saumya Navit

"As a peer-reviewed journal, the Journal of Clinical and Diagnostic Research provides an opportunity to researchers, scientists and budding professionals to explore the developments in the field of medicine and dentistry and their varied specialities, thus extending our view on biological diversities of living species in relation to medicine.
‘Knowledge is treasure of a wise man.’ The free access of this journal provides an immense scope of learning for the both the old and the young in field of medicine and dentistry as well. The multidisciplinary nature of the journal makes it a better platform to absorb all that is being researched and developed. The publication process is systematic and professional. Online submission, publication and peer reviewing makes it a user-friendly journal.
As an experienced dentist and an academician, I proudly recommend this journal to the dental fraternity as a good quality open access platform for rapid communication of their cutting-edge research progress and discovery.
I wish JCDR a great success and I hope that journal will soar higher with the passing time."



Dr Saumya Navit
Professor and Head
Department of Pediatric Dentistry
Saraswati Dental College
Lucknow
On Sep 2018




Dr. Arunava Biswas

"My sincere attachment with JCDR as an author as well as reviewer is a learning experience . Their systematic approach in publication of article in various categories is really praiseworthy.
Their prompt and timely response to review's query and the manner in which they have set the reviewing process helps in extracting the best possible scientific writings for publication.
It's a honour and pride to be a part of the JCDR team. My very best wishes to JCDR and hope it will sparkle up above the sky as a high indexed journal in near future."



Dr. Arunava Biswas
MD, DM (Clinical Pharmacology)
Assistant Professor
Department of Pharmacology
Calcutta National Medical College & Hospital , Kolkata




Dr. C.S. Ramesh Babu
" Journal of Clinical and Diagnostic Research (JCDR) is a multi-specialty medical and dental journal publishing high quality research articles in almost all branches of medicine. The quality of printing of figures and tables is excellent and comparable to any International journal. An added advantage is nominal publication charges and monthly issue of the journal and more chances of an article being accepted for publication. Moreover being a multi-specialty journal an article concerning a particular specialty has a wider reach of readers of other related specialties also. As an author and reviewer for several years I find this Journal most suitable and highly recommend this Journal."
Best regards,
C.S. Ramesh Babu,
Associate Professor of Anatomy,
Muzaffarnagar Medical College,
Muzaffarnagar.
On Aug 2018




Dr. Arundhathi. S
"Journal of Clinical and Diagnostic Research (JCDR) is a reputed peer reviewed journal and is constantly involved in publishing high quality research articles related to medicine. Its been a great pleasure to be associated with this esteemed journal as a reviewer and as an author for a couple of years. The editorial board consists of many dedicated and reputed experts as its members and they are doing an appreciable work in guiding budding researchers. JCDR is doing a commendable job in scientific research by promoting excellent quality research & review articles and case reports & series. The reviewers provide appropriate suggestions that improve the quality of articles. I strongly recommend my fraternity to encourage JCDR by contributing their valuable research work in this widely accepted, user friendly journal. I hope my collaboration with JCDR will continue for a long time".



Dr. Arundhathi. S
MBBS, MD (Pathology),
Sanjay Gandhi institute of trauma and orthopedics,
Bengaluru.
On Aug 2018




Dr. Mamta Gupta,
"It gives me great pleasure to be associated with JCDR, since last 2-3 years. Since then I have authored, co-authored and reviewed about 25 articles in JCDR. I thank JCDR for giving me an opportunity to improve my own skills as an author and a reviewer.
It 's a multispecialty journal, publishing high quality articles. It gives a platform to the authors to publish their research work which can be available for everyone across the globe to read. The best thing about JCDR is that the full articles of all medical specialties are available as pdf/html for reading free of cost or without institutional subscription, which is not there for other journals. For those who have problem in writing manuscript or do statistical work, JCDR comes for their rescue.
The journal has a monthly publication and the articles are published quite fast. In time compared to other journals. The on-line first publication is also a great advantage and facility to review one's own articles before going to print. The response to any query and permission if required, is quite fast; this is quite commendable. I have a very good experience about seeking quick permission for quoting a photograph (Fig.) from a JCDR article for my chapter authored in an E book. I never thought it would be so easy. No hassles.
Reviewing articles is no less a pain staking process and requires in depth perception, knowledge about the topic for review. It requires time and concentration, yet I enjoy doing it. The JCDR website especially for the reviewers is quite user friendly. My suggestions for improving the journal is, more strict review process, so that only high quality articles are published. I find a a good number of articles in Obst. Gynae, hence, a new journal for this specialty titled JCDR-OG can be started. May be a bimonthly or quarterly publication to begin with. Only selected articles should find a place in it.
An yearly reward for the best article authored can also incentivize the authors. Though the process of finding the best article will be not be very easy. I do not know how reviewing process can be improved. If an article is being reviewed by two reviewers, then opinion of one can be communicated to the other or the final opinion of the editor can be communicated to the reviewer if requested for. This will help one’s reviewing skills.
My best wishes to Dr. Hemant Jain and all the editorial staff of JCDR for their untiring efforts to bring out this journal. I strongly recommend medical fraternity to publish their valuable research work in this esteemed journal, JCDR".



Dr. Mamta Gupta
Consultant
(Ex HOD Obs &Gynae, Hindu Rao Hospital and associated NDMC Medical College, Delhi)
Aug 2018




Dr. Rajendra Kumar Ghritlaharey

"I wish to thank Dr. Hemant Jain, Editor-in-Chief Journal of Clinical and Diagnostic Research (JCDR), for asking me to write up few words.
Writing is the representation of language in a textual medium i e; into the words and sentences on paper. Quality medical manuscript writing in particular, demands not only a high-quality research, but also requires accurate and concise communication of findings and conclusions, with adherence to particular journal guidelines. In medical field whether working in teaching, private, or in corporate institution, everyone wants to excel in his / her own field and get recognised by making manuscripts publication.


Authors are the souls of any journal, and deserve much respect. To publish a journal manuscripts are needed from authors. Authors have a great responsibility for producing facts of their work in terms of number and results truthfully and an individual honesty is expected from authors in this regards. Both ways its true "No authors-No manuscripts-No journals" and "No journals–No manuscripts–No authors". Reviewing a manuscript is also a very responsible and important task of any peer-reviewed journal and to be taken seriously. It needs knowledge on the subject, sincerity, honesty and determination. Although the process of reviewing a manuscript is a time consuming task butit is expected to give one's best remarks within the time frame of the journal.
Salient features of the JCDR: It is a biomedical, multidisciplinary (including all medical and dental specialities), e-journal, with wide scope and extensive author support. At the same time, a free text of manuscript is available in HTML and PDF format. There is fast growing authorship and readership with JCDR as this can be judged by the number of articles published in it i e; in Feb 2007 of its first issue, it contained 5 articles only, and now in its recent volume published in April 2011, it contained 67 manuscripts. This e-journal is fulfilling the commitments and objectives sincerely, (as stated by Editor-in-chief in his preface to first edition) i e; to encourage physicians through the internet, especially from the developing countries who witness a spectrum of disease and acquire a wealth of knowledge to publish their experiences to benefit the medical community in patients care. I also feel that many of us have work of substance, newer ideas, adequate clinical materials but poor in medical writing and hesitation to submit the work and need help. JCDR provides authors help in this regards.
Timely publication of journal: Publication of manuscripts and bringing out the issue in time is one of the positive aspects of JCDR and is possible with strong support team in terms of peer reviewers, proof reading, language check, computer operators, etc. This is one of the great reasons for authors to submit their work with JCDR. Another best part of JCDR is "Online first Publications" facilities available for the authors. This facility not only provides the prompt publications of the manuscripts but at the same time also early availability of the manuscripts for the readers.
Indexation and online availability: Indexation transforms the journal in some sense from its local ownership to the worldwide professional community and to the public.JCDR is indexed with Embase & EMbiology, Google Scholar, Index Copernicus, Chemical Abstracts Service, Journal seek Database, Indian Science Abstracts, to name few of them. Manuscriptspublished in JCDR are available on major search engines ie; google, yahoo, msn.
In the era of fast growing newer technologies, and in computer and internet friendly environment the manuscripts preparation, submission, review, revision, etc and all can be done and checked with a click from all corer of the world, at any time. Of course there is always a scope for improvement in every field and none is perfect. To progress, one needs to identify the areas of one's weakness and to strengthen them.
It is well said that "happy beginning is half done" and it fits perfectly with JCDR. It has grown considerably and I feel it has already grown up from its infancy to adolescence, achieving the status of standard online e-journal form Indian continent since its inception in Feb 2007. This had been made possible due to the efforts and the hard work put in it. The way the JCDR is improving with every new volume, with good quality original manuscripts, makes it a quality journal for readers. I must thank and congratulate Dr Hemant Jain, Editor-in-Chief JCDR and his team for their sincere efforts, dedication, and determination for making JCDR a fast growing journal.
Every one of us: authors, reviewers, editors, and publisher are responsible for enhancing the stature of the journal. I wish for a great success for JCDR."



Thanking you
With sincere regards
Dr. Rajendra Kumar Ghritlaharey, M.S., M. Ch., FAIS
Associate Professor,
Department of Paediatric Surgery, Gandhi Medical College & Associated
Kamla Nehru & Hamidia Hospitals Bhopal, Madhya Pradesh 462 001 (India)
E-mail: drrajendrak1@rediffmail.com
On May 11,2011




Dr. Shankar P.R.

"On looking back through my Gmail archives after being requested by the journal to write a short editorial about my experiences of publishing with the Journal of Clinical and Diagnostic Research (JCDR), I came across an e-mail from Dr. Hemant Jain, Editor, in March 2007, which introduced the new electronic journal. The main features of the journal which were outlined in the e-mail were extensive author support, cash rewards, the peer review process, and other salient features of the journal.
Over a span of over four years, we (I and my colleagues) have published around 25 articles in the journal. In this editorial, I plan to briefly discuss my experiences of publishing with JCDR and the strengths of the journal and to finally address the areas for improvement.
My experiences of publishing with JCDR: Overall, my experiences of publishing withJCDR have been positive. The best point about the journal is that it responds to queries from the author. This may seem to be simple and not too much to ask for, but unfortunately, many journals in the subcontinent and from many developing countries do not respond or they respond with a long delay to the queries from the authors 1. The reasons could be many, including lack of optimal secretarial and other support. Another problem with many journals is the slowness of the review process. Editorial processing and peer review can take anywhere between a year to two years with some journals. Also, some journals do not keep the contributors informed about the progress of the review process. Due to the long review process, the articles can lose their relevance and topicality. A major benefit with JCDR is the timeliness and promptness of its response. In Dr Jain's e-mail which was sent to me in 2007, before the introduction of the Pre-publishing system, he had stated that he had received my submission and that he would get back to me within seven days and he did!
Most of the manuscripts are published within 3 to 4 months of their submission if they are found to be suitable after the review process. JCDR is published bimonthly and the accepted articles were usually published in the next issue. Recently, due to the increased volume of the submissions, the review process has become slower and it ?? Section can take from 4 to 6 months for the articles to be reviewed. The journal has an extensive author support system and it has recently introduced a paid expedited review process. The journal also mentions the average time for processing the manuscript under different submission systems - regular submission and expedited review.
Strengths of the journal: The journal has an online first facility in which the accepted manuscripts may be published on the website before being included in a regular issue of the journal. This cuts down the time between their acceptance and the publication. The journal is indexed in many databases, though not in PubMed. The editorial board should now take steps to index the journal in PubMed. The journal has a system of notifying readers through e-mail when a new issue is released. Also, the articles are available in both the HTML and the PDF formats. I especially like the new and colorful page format of the journal. Also, the access statistics of the articles are available. The prepublication and the manuscript tracking system are also helpful for the authors.
Areas for improvement: In certain cases, I felt that the peer review process of the manuscripts was not up to international standards and that it should be strengthened. Also, the number of manuscripts in an issue is high and it may be difficult for readers to go through all of them. The journal can consider tightening of the peer review process and increasing the quality standards for the acceptance of the manuscripts. I faced occasional problems with the online manuscript submission (Pre-publishing) system, which have to be addressed.
Overall, the publishing process with JCDR has been smooth, quick and relatively hassle free and I can recommend other authors to consider the journal as an outlet for their work."



Dr. P. Ravi Shankar
KIST Medical College, P.O. Box 14142, Kathmandu, Nepal.
E-mail: ravi.dr.shankar@gmail.com
On April 2011

Important Notice

Reviews
Year : 2012 | Month : May | Volume : 6 | Issue : 4 | Page : 740 - 744

Neurotoxic Effects of Fluoride in Endemic Skeletal Fluorosis and in Experimental Chronic Fluoride Toxicity

Shivarajashankara Y.M., Shivashankara A.R.

1. Professor of Biochemistry, KVG Medical College, Sullia. 2. Associate Professor of Biochemistry, Father Muller Medical College, Mangalore, Karnataka, India.

Correspondence Address :
Dr. Shivashankara A.R. PhD.,
Associate Professor of Biochemistry,
Father Muller Medical College, Mangalore, India - 575002,
Phone: +918242238255; +919880146133.
E-mail: sramachandrayya@gmail.com.

Abstract

Fluoride has a significant impact on the human health. Traces of fluoride are beneficial in preventing dental caries and osteoporosis, but the long-term intake of high levels of fluoride, mainly via drinking water, causes detrimental effects. High levels of fluoride cause dental and skeletal fluorosis and even the soft tissues are not spared from fluoride toxicity. Fluoride can cross the blood brain barrier and it can cause adverse effects on the brain cell architecture, metabolism, enzymes, the oxidantantioxidant status and on neurotransmitters and overall adverse effects on the mental functions. Fluoride induces the generation of free radicals, it increases lipid peroxidation, it impairs the antioxidants, it inhibits the key enzymes of the metabolic pathways, it impairs energy generation, and it inhibits protein synthesis. Excitoxicity, which has been proposed as the major mechanism in the neurotoxic manifestations of fluoride, needs a detailed and critical evaluation.

Keywords

Brain, Excitotoxicity, Fluoride, Fluorosis, Neurotoxicity

Introduction
Fluorine is a highly electronegative trace element which is the 13th most abundant element in the earth’s crust (1). It is highly reactive and it occurs ubiquitously as fluorides in nature. Extensive research has been carried out on the chemistry and the biology of fluoride, and on its impact on the human health. Fluoride, in trace concentrations, is required to prevent dental caries, while the longterm consumption of excess fluoride, mainly via drinking water, leads to a spectrum of toxic manifestations which are referred to as fluorosis (1). Fluorosis, as a global public health problem, has been receiving wide and deserving attention in recent years. Fluoride has been shown to be toxic, not only to the skeletal tissues, but also to the non-skeletal tissues such as the brain, liver, pancreas, endocrines and the kidney (1),(2).

Fluoride can cross the blood brain barrier (3), and it can cause adverse effects on the brain cell architecture, metabolism, enzymes, the oxidant-antioxidant status and on neurotransmitters and overall adverse effects on the mental functions (4),(5),(6),(7). In this review, we have focussed on the effects of chronic fluoride toxicity on the structural and functional aspects of the brain in experimental animals and on the neurotoxicity of fluoride in endemic skeletal fluorosis.

The Environmenta l Sources and the Levels of Fluoride
Human beings are exposed to fluoride from various sources such as ground water, food ,air, drugs, cosmetics, tooth paste and dental applications (1),(8). The effects of fluorides on the human health stem largely from dissolved fluorides which are present in ground water and drinking water contributes to more than 60% of the total fluoride intake (1),(9). In rocks and soil, fluoride occurs in a wide variety of minerals, which include fluorspar (CaF2), cryolite (3 NaF AlF2), apatites ( 3 Ca2 [PO4] 2 Ca[F] 2), mica and hornblende (1). Volcanic rocks and salt deposits of marine origin also contain significant amounts of fluoride. The fluoride levels are found to be high in soft, alkaline, calcium-deficient waters, and in interior arid areas. A water fluoride level of 0.5-1.0 ppm is considered to be optimum, which is required to prevent dental caries, above which (>1.0 ppm ) clinical manifestations of dental and skeletal fluorosis are seen (1),(9).

The fluoride content of the top soil is increased by the addition of phosphate fertilizers, pesticides and irrigation water and by the deposition of gaseous, particulate emissions. The fluoride content of foods is significantly affected by the fluoride content in the water which is used for growing crops and for processing foods (1). Tea, marine fish, animal tissues and certain vegetations are found to contain highly significant amounts of fluoride. The fluorides in air originate from the dusts of fluoride containing soils, gaseous industrial wastes, the domestic burning of coal fires and from gases which are emitted in areas of volcanic activity. Many of the lakes of the Rift Valley system with high volcanic activity are found to have fluoride concentrations upto 2800 ppm (1),(10).

Fluoride and the Nervous System
1. The Neurological Manifestations of Skeletal Fluorosis
The most obvious and the earliest clinical manifestation of fluoride toxicity is dental fluorosis, which can develop in children, due to the intake of high levels of fluoride during the period of tooth development, and clinically, it is the marker of fluoride toxicity in the first six years of life (1). The fluoride accumulation in bones over a long time results in skeletal fluorosis and its early symptoms include stiffness and pain in the joints. Crippling skeletal fluorosis is associated with osteosclerosis, calcification of the tendons and the ligaments, and bone deformities (1),(11),(12),(13). Fluorosis exhibits neurological problems such as a tingling sensation in the fingers and toes, nervousness and depression. In the advanced stages of fluorosis, neurological manifestations such as paralysis of the limbs, vertigo, spasticity in the extremities, and impaired mental acuity, are observed in human beings (1),(12),(13).

2. Fluoride Accumulation in the Brain
Fluoride is known to enter the brain and the blood brain barrier fails to exclude fluoride from the nervous tissue (3). The transport of fluoride through the blood brain barrier is an active transport system which is similar to that of other halogens and ionic substances, and the normal CSF/blood fluoride ratio is less than 1.0 (14).

Mullenix et al., observed an accumulation of fluoride in the important regions of the brain, especially in the hippocampus (mean 0.993 ppm F at 125 ppm water fluoride during weanling), which was found to increase as the fluoride levels in the drinking water increased (4). The administration of NaF (20mg/kg, 14 days) caused an increased accumulation of fluoride in the brain of fluoride-treated rats (0.293 μg/g dry tissue; + 8%) , as compared to that in the control rats (7). Maternal exposure to 100 ppm fluoride in drinking water, resulted in fluoride accumulation (upto 2.14 μg/ g tissue) in the brain of young rats when compared to controls (0.27 – 0.64 μg/ g tissue) (15).

3. Fluoride and the Brain Cell Health
Fluoride is toxic to the brain and chronic fluoride intoxication causes abnormalities in the brain cell architecture. There are many reports of histological abnormalities in the brain tissue of animals which were exposed to high levels of fluoride directly or during the foetal and weanling stages via the mother (4),(5),(16),(17),(18). The passage of fluoride thorough the placenta of mothers with chronic fluorosis and its accumulation within the brain of the foetus is shown to have an adverse impact on brain development. Du et al. studied the brains of foetuses from endemic fluorosis areas, that were aborted therapeutically at the 5th–8th month of gestation, in comparison to the foetuses from non-endemic areas (16). They observed reductions in the mean volume of the neurons, the numerical density of the volume, volume density, and in the surface density of the mitochondria in the foetuses from endemic fluorosis areas (16). Offspring rats which were exposed to high fluoride (45 ppm) and low iodine for 90 days exhibited neurotoxic changes in brain, which were indicated by neurons with pycknosed nuclei, decreased Nissl substance, and elongated dendrites (19). The rats which were exposed to high fluoride and low iodine in water (100 ppm F, from one month to 20 months), showed considerable DNA damage of up to 92%, in the brain cells (20).

There have been attempts to assess the morphological changes in the various subregions of the brain in fluoride-treated animals. In a study which was done by our group, rats were exposed to 30 or 100 ppm fluoride (as NaF) in drinking water during their foetal (maternal exposure), weanling (maternal exposure), and post weaning stages of life until the age of ten weeks. Young rats which were exposed to 30 ppm fluoride did not show any notable alterations in the brain histology, whereas the rats which were exposed to 100 ppm fluoride showed significant neurodegenerative changes in the hippocampus, amygdala, motor cortex and the cerebellum. The changes included a decrease in the size and the number of the neurons in all the regions of brain, a decrease in the number of the Purkinje cells in the cerebellum, and signs of chromatolysis and gliosis in the motor cortex (5). Subcutaneous injections of sodium fluoride ( 5-50 mg/ml/kg/day for 15 weeks) in rabbits caused loss of the molecular layer and the glial cell layer in the brain tissues; chromatolysis and a ballooned appearance of the Purkinje neurons; vacuolization in the perikaryon; and the presence of spheroid bodies in the neuroplasm (18).

The chronic administration of fluoride as NaF or AlF3 in the drinking water (1 ppm F, 52 weeks) resulted in distinct morphological alterations in the brain of rats ; in the reduction of the neuronal density,chromatin clumping, enhanced protein staining, pyknosis and vacuolation. The presence of ghost-like cells in the left hemisphere was more prominent in the AlF3 –treated group than in the NaFtreated group (21).The fluoride toxicity also resulted in abnormal alterations in the cerebrovasculature (21).

4. Effect of Fluoride on the Metabolism in the Brain
Fluoride is an inhibitor of many enzymes which require divalent cations as cofactors. The enzymes which are inhibited by fluoride are concerned with energy metabolism, the metabolism of proteins and amino acids, and the scavenging of free radicals. Hence, fluoride is considered as a metabolic poison, and it is known to alter the metabolic pathways in tissues such as the liver, muscle and the brain (22),(23),(24),(25),(26). With regards to the effect of fluoride on the metabolism in the brain, studies have shown that fluoride (as NaF) impairs the activities of the enzymes which are concerned with the metabolism of lipids, proteins and nucleic acids, and transmission of the nerve impulse (7), (27), (28), (29), (30), (31). The exposure to fluoride (30 or 100 ppm for 3–7 months) caused changes in the membrane lipids in the brain (30). Sodium fluoride administration decreased the contents of phosphatidyl ethanolamine, phosphatidyl choline and phosphatidyl serine, and it increased the ubiquinone levels in the brain cell membrane (30),(31). Subcutaneous injections of sodium fluoride (5-50 mg/kg/day, 100 days) increased the contents of the total lipids, phospholipids and the triglycerides in the brain of rabbits (27). With regards to the effect of chronic fluoride intoxication on the enzymes which were concerned with energy metabolism, the fluoride administration (NaF at a dose of 20 mg/kg/day, 14 days) in mice reduced the activities of lactate dehydrogenase (LDH), succinate dehydrogenase (SDH), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and creatine kinase (CK) in the brain (7).

Fluoride inhibits the activities of the enzymes which are concerned with membrane function and nerve impulse transmission. Sodium fluoride reduced the activities of sodium-potassium ATPase, magnesium ATPase, calcium ATPase and acetyl cholinesterase in the brain (7). The administration of 5 or 50 ppm fluoride for 6 months to rats resulted in the decreased activities of acetylcholinesterase and butyrylcholinesterase in the brain tissue, and the effect was more pronounced with 5 ppm F (32). Contrary to this observation, maternal exposure to high levels of fluoride (5,15,50 ppm F) and continuation of the same treatment after birth till 80 days, resulted in an elevated activity of acetylcholinesterase in the cerebral synaptic membranes (33). Long et al., observed a significant reduction in the number of nicotinic acetylcholine receptors in the brain of rats which were exposed to sodium fluoride (34).

The inhibitory effect of fluoride on the synthesis of nucleic acids and protein in the brain, has been reported by few authors . The oral administration of sodium fluoride (NaF, 6 and 12 mg/kg body weight/day, for 30 days) caused a significant, dose-dependent reduction in the DNA, RNA, and the protein contents in the cerebral hemisphere, cerebellum, and in the medulla oblongata of the brain in mice. After the withdrawal of the treatment for 30 days, a partial but significant amelioration occurred (35). The fluoride intoxication in rabbits resulted in decreased contents of the total, soluble and the basic proteins, with an increase in the free amino acids in the brain (28).

5. Fluoride and the Antioxidant System of the Brain
Fluoride is known to induce oxidative stress and to impair the functioning of antioxidants in the brain. Various studies on experimental animals have observed an increased lipid peroxidation and decreased or increased levels of antioxidants in the brain tissue, on exposure to sodium fluoride. The fluoride administration (100 ppm, in drinking water for 3 months) in adult rats resulted in increased malondialdehyde (MDA), glutathione (GSH), glutathione S-transferase (GST), glutathione peroxidase (GSH-Px) and ascorbic acid in the brain (36). The administration of 5 or 50 ppm fluoride for 6 months to rats resulted in a decreased total antioxidant capacity and in increased MDA, in the brain (32). Krechniak and Inkielewicz observed a strong positive correlation of the brain fluoride content with the degree of oxidative stress; the brain fluoride content showed a positive correlation with MDA and the protein carbonyls,and a negative correlation with GSH and GSH-Px in the brain of rats which were subjected to chronic fluoride toxicity 37). Fluoride (at a dose of 20 mg/kg/day, 14 days) increased the activity of the prooxidant enzyme, xanthine oxidase and it reduced the activities of the antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT) and GST in the brain of mice (7).

The maternal exposure to fluoride and fluoride intoxication at the early stages of life is shown to cause more pronounced effects on the oxidant-antioxidant status in the brain than the fluoride exposure at a later stage (adolescent/adult) of life (6),(15), (36), (38).Rats which were exposed to 100 ppm fluoride (as NaF) in drinking water during the last one week of intrauterine life (through maternal exposure) and which were then exposed up to ten weeks after birth, showed elevated MDA and GSH-Px levels, and decreased levels of total glutathione, GSH and ascorbic acid in the brain (6). The maternal exposure to 100 ppm fluoride in drinking water, resulted in increased lipid peroxidation, and in decreased levels of SOD, CAT, GSH-Px, GST and GSH in the brain of offspring rats (15). Basha et al., carried out a study to assess the effect of fluoride (100, 200 ppm in drinking water) on the oxidative stress in the brain, for three generations of rats ; they observed that the fluorideinduced increase in the lipid peroxidation and the decreases in the antioxidants were more pronounced with the second and third generations as compared to the first generation of the rats (38).

Fluoride, in combination with arsenic, is shown to have immense effect on the oxidant–antioxidant status in brain. Fluoride and arsenic singly or in combination caused increased levels of dehydroascorbic acid and lipid peroxides, and a decrease in the SOD, CAT, GSH-Px, GSH and the ascorbic acid levels in the brain of rats; the effect was more pronounced with a combination of fluoride and arsenic than with these compounds independently (39).

6. Fluoride and Mental Functions
There have been reports of decreased mental acuity and impaired mental functions, both in experimental animals which were subjected to fluoride toxicity, and in fluorotic children. In 1937, Kaj Roholm , the pioneer of fluoride research, published his findings on skeletal fluorosis with excessive tiredness, sleepiness, indisposition, headache, and giddiness in cryolite workers. The first case of skeletal fluorosis was reported in Andhra Pradesh in India in 1937 (40),(41). After this, many researchers have reported neurological manifestations such as tingling sensations, loss of the sensations of pain, temperature and touch, altered reflexes, and loss of the sphincter control in skeletal fluorosis patients (12),(40),(41). Many studies have shown that the children in fluorosis endemic areas were prone to mental retardation and that their IQ was low. Researchers, mainly from China, have reported that the IQ of children from high-fluoride, endemic fluorosis areas was significantly lower as compared to that of children from areas with less than 1 ppm fluoride in drinking water (42), (43), (44), (45), (46). Higher drinking water fluoride levels were found to be associated with higher rates of mental retardation (IQ <70) and borderline intelligence (IQ 70-79). The incidence of Down’s syndrome was shown to be higher among births to younger mothers in high-fluoride areas (46). High levels of fluoride in drinking water depressed the learning and memory abilities of the brain, and caused behavioural deficits even in rats and mice (4),(17),(32), (47),(48).

7. Mechanisms of the Neurotoxicity of Fluoride
7.1. Fluoride Induces Oxidative Stress
Fluoride exerts its toxic effects on the brain by multiple mechanisms; the primary phenomenon which is involved in the neurotoxicity of fluoride appears to be oxidative stress. Fluoride is known to induce the generation of free radicals and to result in the consequent oxidative stress (49),(50). Because of its high electronegativity, F–forms strong hydrogen bonds, especially with the –OH and –NH moieties in biomolecules, and it has a potent ability to form stable complexes with polyvalent metal cations like Al3+, Fe3+, and Mg2+ (49),(50).

We observed elevated MDA, GSH-Px and vitamin C levels, and reduced levels of GSH, uric acid and SOD in the blood of children with endemic skeletal fluorosis (51). While the elevated MDA levels which indicate increased lipid peroxidation, is a universal finding in the animal studies on oxidative stress in fluorosis, varied observations have been made on the changes in the antioxidants in blood and tissues, including the brain. The elevations in the antioxidants have been attributed to an adaptive response and the protective mechanism of the tissue to fluoride-induced oxidative stress, while the decreased levels of the antioxidants have been attributed to their depletion on combating the reactive oxygen species which are generated in chronic fluoride toxicity (15), (32), (36), (38), (49), (52). An in vitro study has shown that fluoride inhibits the antioxidant enzyme, SOD (53).

7.2. Fluoride Inhibits Enzymes and Alters Metabolic Pathways The biotoxicity of the fluoride ions results mainly from their inhibitory effect on the activity of many enzymes, mostly of ATP production and those which synthesize protein and DNA (22), (23), (24), (25), (26). This is associated with the high chemical activity of the F ion and its affinity to Ca2+ and Mg2+, which catalyze a number of enzymatic reactions. Few such enzymes include enolase, ATPases, cholinesterases, arginase, ACP, SDH, esterases, isocitrate dehydrogenase, phosphatases and aconitase (22), (23), (24), (25), (26). Inhibitions of the enzymes of glycolysis, the citric acid cycle and the electron transport chain have an adverse impact on the energy generation in the mitochondria. Studies have shown that the treatment with sodium fluoride decreases the activities of LDH, SDH, ALT, AST, CK, ATPases and actetyl cholinesterase in the brain of fluorideintoxicated animals (7),(32). These findings indicate that fluoride impairs the energy generation, the membrane function, the amino acid metabolism and the nerve impulse transmission. Fluoride is shown to alter the metabolism of glucose, lipids and amino acids in various tissues (27),(28),(54).

7.3. Fluoride Inhibits the Synthesis Of DNA, RNA And Protein, And Induces DNA Damage
In vitro studies have revealed that NaF affects the cellular protein synthesis by impairing the peptide chain initiation (55). The incubation of He La cells with NaF resulted in the inhibition of protein synthesis, disaggregation of the polyribosomes, accumulation of the 80 S ribosomes and in the decrease of the free ribosomal subunits. After the removal of NaF, the normal level of the free ribosomal subunits was restored at the expense of a random dissociation of the ribosomes (55). In vitro studies have also shown that fluoride inhibits the incorporation of amino acids into a polypeptide chain (55). The decreased levels of proteins, DNA and RNA, and the increased content of the free amino acids in the brain of animals which were exposed to chronic fluoride toxicity substantiated the findings of the in vitro studies (28),(35). Fluoride is also known to induce DNA damage and apoptosis in the brain (20).

7.4. Fluoride acts synergistically with aluminium and mediates the effects through the G-protein–coupled receptors
Aluminium and fluoride have a close association in drinking water sources, the environment and in cooking utensils, where they form aluminofluoride complexes. Aluminium reinforces fluoride’s stimulation of the G protein-coupled receptor-mediated signal transduction (56). Brain cells have their second messenger systems mediated through the activation of the G-protein coupled receptors, which are known to be influenced by fluoride alone or in combination with aluminium. These mechanisms include, activation of adenylate cyclase, increase in the levels of cyclic AMP; activation of protein kinase C and the subsequent activation of mitogenactivated protein kinase C(MAPK) and the phosphoinositide second messenger system (56), (57).

7.5. Excitotoxicity as the central mechanism in the neurotoxicity of fluoride
Excitotoxicity by microglial activation is proposed to play a central role in the neurotoxicity of fluoride (58). It is a universal mechanism which is known to be involved in many neurological disoders, and in the neurotoxic manifestations of many compounds and heavy metals. Excitoxicity is caused due to the overstimulation of the glutamate receptors in the neurons, leading to activation of the microglia (the immune cells of the brain) which release cytokines and other immune factors, finally causing brain cell death. The glutamate receptors get overstimulated because of the accumulated glutamate as a result of the impaired glutamate transporters of the brain. The cascade of excitoxicity is mediated through the reactive oxygen species (ROS) and the reactive nitrogen species (RNS), which may also be the causative factors in excitoxicity (58).

The theory of excitotoxicity as a central mechanism in the neurotoxicity of fluoride has been proposed by Blaylock , and circumstantial evidences are available to propose the excitotoxicity in fluorosis (58). Many animal studies have shown that fluoride accumulates in the areas of the hippocampus, dentate gyrus, and in the superficial areas of the amygdala, cortex and the cerebellum, leading to histopathological abnormalities (4),(5),(18),(19). These areas have an abundance of glutamate receptors. Fluoride is known to induce the generation of ROS and RNS in the brain (7), (36), which are the principal mediators of the excitoxicity. As has been explained under the mechanisms, 1 and 2, fluoride impairs the energy metabolism and the mitochondrial functions. Fluoride alone, or in combination with aluminium, triggers a cascade of molecular events through the G protein-coupled receptors, the second messenger systems, –c AMP, c GMP and the phosphoinositides and through the activation of protein kinase C, thereby stimulating the processes which are involved in the microglial activation (58). However, there is a lack of direct experimental evidences which shows that fluoride specifically activates the microglia, and that fluoride stimulates the glutamate receptors.

Summary
Fluoride is a neurotoxin and it makes a serious adverse impact on the developing brain. Impaired mental functions are observed among children in endemic fluorosis areas and in experimental animals with fluoride-induced neurotoxicity. Fluoride induces the generation of free radicals, it increases lipid peroxidation, it impairs antioxidants, it inhibits the key enzymes of the metabolic pathways, it impairs energy generation, and it inhibits protein synthesis. The animal experiments which were done on chronic fluoride toxicity have reported varied findings, which might be due to the differences in the dose, duration and the mode of the fluoride administration, the animal species which was used, and the organspecific metabolic responses. Excitotoxicity, which is proposed as the central mechanism in the neurotoxic effects of fluoride, needs critical evaluation by mechanistic studies and there is a need for extensive research on the amelioration of the fluoride-induced pathology of the brain.

Acknowledgement

The authors wish to dedicate this review paper to their mentors, Professor P.Gopalakrishna Bhat (Professor of Biochemistry, KMC Manipal) and Professor S. Hanumanth Rao (Former Professor of Biochemistry, MR Medical College, Gulbarga), two inspiring teachers. We gratefully remember the fluorosis patients from Kheru Nayak Thanda, Gulbarga, on whom our first research paper was published.

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DOI and Others

ID: JCDR/2012/3590:0055

Date of Submission: Nov 10, 2011
Date of peer review: Feb 06, 2012
Date of acceptance: Apr 01, 2012
Date of Publishing: May 31, 2012

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