Jennifer Anne Luke
Fluoride Action Network
Posted December 19, 2011
Jennifer Anne Luke, 1997
A dissertation submitted to the School of Biological Sciences, University of Surrey, in fulfillment of the requirements for the Degree of Doctor of Philosophy.
Excerpts from pages: 1-9; 51-53; 167-177
The Effect of Fluoride on the Physiology of the Pineal Gland
The purpose was to discover whether fluoride (F) accumulates in the pineal gland and thereby affects pineal physiology during early development. The [F] of 11 aged human pineals and corresponding muscle were determined using the F-electrode following HMDS/acid diffusion. The mean [F] of pineal gland was significantly higher (p Chapter 1 Background Information
In this study I attempted to discover whether fluoride (F) has pathophysiological effects on the pineal gland: a feasible proposition if F accumulates in the pineal and can thereby influence its physiology. The pineal gland, or seat of the soul as it is colloquially called, is situated near the anatomical centre of the brain. It is an integral part of the central nervous system (CNS). Fluoride metabolism in the CNS has not been systematically studied. It is generally believed that F has no effect on the CNS because it is excluded from brain by the blood-brain barrier (Whitford et al, 1979). Whole brain has a low F-content like normal soft tissues elsewhere in the body.
It is remarkable that the pineal gland has never been analysed separately for F because it has several features which suggest that it could accumulate F. It has the highest calcium concentration of any normal soft tissue in the body because it calcifies physiologically in the form of hydroxyapatite (HA). It has a high metabolic activity coupled with a very profuse blood supply: two factors favouring the deposition of F in mineralizing tissues. The fact that the pineal is outside the blood-brain barrier suggests that pineal HA could sequester F from the bloodstream if it has the same strong affinity for F as HA in the other mineralizing tissues.
The intensity of the toxic effects of most drugs depends upon their concentration at the site of action. The mineralizing tissues (bone and teeth) accumulate high concentrations of F and are the first to show toxic reactions to F. Hence, their reactions to F have been especially well studied. If F accumulates in the pineal gland, then this points to a gap in our knowledge about whether or not F affects pineal physiology. It was the lack of knowledge in this area that prompted my study.
Fluoride and The Pineal Gland
Children are now exposed to more F than ever before. Fluorides are the cornerstone of all caries preventative programs. The substantial reduction in the incidence of dental caries in the western world over the past fifty years has been largely attributed to the access to fluoridated water supplies and the increased exposure to F in dental products. The fluoridation of water supplies is an important public health measure. It is endorsed by the WHO, the European Union directives, the Royal College of Physicians, the Royal College of General Practitioners, the BMA, and the medical and dental professions (Samuels, 1993).
Despite the endorsements, the prophylactic use of F in dentistry has been a controversial subject for decades. One recent study reported an increase in osteosarcomas in male F3441N rats which had received drinking water containing 100-175 mg NaF/L (45-79 mg F/L) for two years (NTP, 1990). Following this report, three critical bodies analysed the public health benefits and risks from chronic F-exposure by reviewing the evidence from human epidemiological studies of the relationship between cancer and water fluoridation and also carcinogenicity studies in rodents. They unanimously agreed that F is safe and effective if used appropriately. The use of F is not associated with an increased cancer risk in humans. Dental fluorosis is the only adverse effect associated with the chronic ingestion of relatively low F-levels (Kaminsky et al, 1990; USPHS, 1991; NRC, 1993). Further research is required on the effects of F on the reproductive system in animals and humans (USPHS, 1991).
Dental fluorosis (defective, hypomineralized enamel) occurs when excessive amounts of F reach the growing tooth during its developmental stages. The manifestations of fluorosis range from barely noticeable opacities to severely pitted teeth. The greater the F-exposure during tooth formation the greater is the likelihood of dental fluorosis developing and the more severe is the pathology. The F-concentration at which fluorosis becomes apparent in a population corresponds to a daily intake of about 0.1 mg F/kg body weight (BW) up to the age of 12 years although there is no firm consensus on this issue. In fact, a high prevalence and severity of dental fluorosis was reported in populations with an estimated daily F-intake of less than 0.03 mg F/kg BW (Blum et al, 1987).
The so-called ‘optimal’ concentration of F in community water is defined as the concentration of F which gives maximum caries reduction and causes minimum dental fluorosis, i.e., between 0.7 and 1.2 mg/L depending on the mean ambient temperature. At the time of Dean’s original studies, there was a 10-12 percent prevalence of mild dental fluorosis in children in the 1 mg F/L areas (Dean et al, 1941, 1942). This was ‘accepted’ in return for the benefits in caries reduction: a classic public health trade-off. In the 1930s and 1940s, virtually the only source of F was in the drinking water. Today, F is ubiquitous in the environment which means that man’s daily F-intake comes from several sources besides tap water.
Systemic F-exposure to children has increased (Leverett, 1991). Mild dental fluorosis is now more common than one would predict on the basis of Dean’s findings in the late 1930s and early 1940s: in fluoridated and non-fluoridated communities (Leverett 1986; Pendrys and Stamm, 1990; USPHS, 1991). Several recent studies report prevalence rates in the 20 and 80 percent range in areas with fluoridated water (Levy, 1994). The prevalence of 0.9 percent (recorded in the pre-fluoride days) in areas containing less than 0.4 mg F/L in the water has increased to 6.6 percent (USPHS, 1991). The prevalence of moderate to severe dental fluorosis has increased USPHS, 1991; Lalumandier and Rozier, 1995). The increased prevalence of dental fluorosis is causing concern within the scientific community because it is an early sign of F-toxicity and evidence that some children are now getting more F than is good for them. The issue has the potential to become a significant dental health problem.
Of all the tissues, the developing enamel organ is assumed to be most sensitive to the toxic effects of F. It contains significantly higher concentrations of F and calcium than other soft tissues. The enamel organs of 9-day-old rats contained significantly higher F levels than corresponding soft tissue (0.14 vs. 0.015 mg/kg). Following oral administration of F to the rat pups (0.5 mg/kg BW), the [F] of the enamel organ reached peak values (0.19 mg/kg) in 30 minutes. The enamel organ may be relatively sensitive to increased systemic F-intake because it accumulates F (Bawden et al, 1992).
Although the exact mechanism responsible for enamel fluorosis is not known, F may have specific effects on the normal activity of ameloblasts, developing enamel matrix and proteolytic activity in the maturing enamel (DenBesten and Thariani, 1992). The transition/early-maturation stage of amelogenesis is most susceptible to the effects of an increased plasma F-concentration. The aesthetically important maxillary central incisors are most vulnerable to F at 22-26 months (Evans and Stamm, 1991).
Alongside the calcification in the developing enamel organ, calcification is also occurring in the child’s pineal. It is a normal physiological process. A complex series of enzymatic reactions within the pinealocytes converts the essential amino acid, tryptophan, to a whole family of indoles. The main pineal hormone is melatonin (MT). For some reason, young children have the highest levels of plasma MT. They also have higher plasma F levels (recommended from a dental perspective) than they did 50 years ago. An increasing number of children suffer from mild dental fluorosis: evidence that they received too much F during the first few years of life. If F accumulates in the pineal gland during early childhood, it could affect pineal indole metabolism. In much the same way that high local concentrations of F in enamel organ and bone affect the metabolism of ameloblasts and osteoblasts.
If F influences the high pineal MT output during early development, then the functions of the pineal may also be compromised (given that MT is the main mediator of pineal function). One putative function of the pineal is its involvement in the onset of puberty. If F compromises pineal function by altering the high rate of synthesis of MT during childhood, does this manifest as an alteration in the timing of puberty?
Although the extrapolation of results from animal studies to the human situation is difficult, this project may identify a potential health risk to humans. Therefore, the results will either affirm the safety of the extensive use of F in dentistry or suggest that harmful effects on human health have already occurred: either way, this investigation is worthwhile.
Professor Paul Connett: Your Toxic Tap Water
1.2 Review of the Literature
To the best of my knowledge, the Newburgh-Kingston study is the only reference on the effect of F on the timing of puberty in humans. It is the largest, most ambitious paediatric survey carried out to demonstrate the safety of water fluoridation. The New York State Department of Health initiated the study in 1944 because they realized that there would ultimately be a need for a long-term evaluation of any possible systemic effects as well as the dental changes from drinking fluoridated water over a long period of time.
\Similar groups of children were selected for long-term observation from Newburgh (fluoridated to 1.0 to 1.2 mg/L in 1945) and Kingston (essentially F-free for the duration of the study). Newburgh and Kingston were chosen because they were well-matched: both were situated on the Hudson River about 35 miles apart with similar upland reservoir water supplies; both had populations of about 30,000 with similar demographic characteristics, social and economic conditions, levels of dental care, etc. In Newburgh, out of 817 children (aged from birth to nine years) who were selected in 1945, 500 were examined in 1954-1955; in Kingston, out of 711 children who were selected in 1945, 405 were examined in 1954-1955.
The medical and dental examinations began in 1944, and were repeated periodically until 1955. An assessment of any possible systemic effects arising from the consumption of fluoridated water was made by comparing the growth, development and the prevalence of specific conditions in the two groups of children as disclosed by their medical histories, physical examinations, and laboratory and radiological evidence. The age of onset of menstruation in girls was used as an index of the rate of sexual maturation.
At the end of ten years, the investigators reported no adverse systemic effects from drinking fluoridated water because no significant differences were found between the results from the two groups. The average age of first menarche was earlier among girls in Newburgh than those in Kingston: 12 years vs. 12 years and 5 months respectively (Schlesinger et al, 1956). Although this difference was not considered important, it does suggest an association between the use of fluoridated drinking water and an earlier onset of sexual maturation in girls. The Newburgh girls had not had a lifelong use of fluoridated water. For the first two years or so, they received unfluoridated water. Furthermore, their only source of F was from the drinking water.
1.3 Sources of Fluoride
The normal daily F-intake is negligible (less than 0.01 mg) during the first few months of human life, because human breast milk contains merely a trace of F (6 to 12 ng/ml): regardless of the F-intake to the nursing mother. Ekstrand and co-workers (1981) analysed plasma and milk samples from five nursing mothers after they had taken an oral dose of 1.5 mg F. There was an immediate ten-fold increase in the [F] of plasma (70-86 ng/ml) within 30 minutes of dosing but the [F] of breast milk remained constant throughout the day (2-8 ng/ml). The mean F-concentrations of human breast milk were 8.9 and 5.0 ng/ml from nursing mothers living in 1.7 and 0.2 mg Fit areas respectively (Esala et al, 1982); 6.8 ± 0.4 and 5.3 ± 0.4 ng/ml (± SEM) from nursing mothers living in 1.0 and 0.2 mg Fit areas respectively (Spak et al, 1983).
The reason for the limited transfer of F from plasma to breast milk is unknown. It has been suggested that the physiological plasma-milk barrier actively protects the newborn from the toxic effects of F (Ekstrand et al, 1981). Cow’s milk, like human milk, contains low levels of F (0.017 mg/L) even when F is added to the cow’s food or drinking water (McClure, 1949). Breast-fed infants (or infants bottle-fed with cow’s milk) are in negative F-balance: more F is excreted in the urine than is ingested in the diet. During the period of breast feeding, F (deposited in foetal bone during pregnancy) is mobilized and released into the extracellular fluids and subsequently excreted into urine. Therefore, early human development has always occurred in a virtually F-free milieu even in the high-F areas: a phenomenon which lasts until the age of weaning and the introduction of solid foods.
In contrast, the F-intake to bottle-fed infants living in fluoridated areas depends upon the [F] of. a) the water used to reconstitute the feed; b) the powdered formula-feed itself. Bottle-fed infants in fluoridated areas can receive 1.1 mg F from day 1: 150-200 times more F per day than breast-fed infants, i.e., 1100 vs. 5-10 j.tg/day (Ekstrand, 1989). The normal pharmacokinetics of F during infancy is reversed. Bottle-fed infants in fluoridated areas retain more than 50% of the ingested F-dose in the mineralizing tissues (Ekstrand et al, 1984; 1994).
Man’s daily intake of F from food is low. Fresh, unprepared vegetables, fruits, pulses, roots, nuts, etc., rarely contain more than 0.2 to 0.3 mg F/kg (WHO, 1984). Most plant species have a limited capacity to absorb F from the soil even when F-containing fertilisers are applied (Davison, 1984). The flesh of meat, poultry and fish, (free from bone), contains low levels of F because virtually all F in animals occurs in their bones and teeth. The skin and bones of tinned salmon and sardines contain 8 and 500 mg F/kg respectively because the fish are exposed to relatively high levels of F (1.2-1.4 mg/L) in sea-water (Jenkins, 1990).
Chapter 2 – Aims and Objectives
1. The purpose of the first experiment was to discover whether F accumulates in the human pineal gland. The objectives were to determine:
a) The [F] of human pineal gland and corresponding muscle and bone so that the pineal [F] could be compared to that of muscle and bone.
b) The [Ca] of human pineal gland so that pineal [Ca] could be correlated with pineal [F], and an estimation made of the amount of hydroxyapatite (HA) in the pineal.
2. The purpose of the second experiment was to discover whether F affects pineal physiology: specifically, its ability to synthesize melatonin (MT). The aim was to set up a controlled longitudinal study of the effects of F on the pineal output of MT during the transition from prepubescence through puberty into young adulthood using the Mongolian gerbil (Meriones unguiculatus) as the experimental animal model. The levels of urinary 6-suiphatoxymelatonin, aMT6s, were used as an index of pineal MT synthesis.
The objectives were:
a) To collect urine from two groups of gerbils, high-F (HF) and low-F (LF), at 7, 9, 11Y2 and 16 weeks of age at 3-h intervals over 48-h for the subsequent measurement of the levels of urinary aMT6s. The levels of urinary aMT6s were determined using radioimmunoassay (RIA).
b) To validate the RIA for urinary aMT6s currently in use in the laboratory for use with gerbil urine.
c) To demonstrate that the amount of MT synthesized by the gerbil pineal reflects the excretion rate of aMT6s in gerbil urine in 16-week-old gerbils. The aims were to determine the gerbil pineal MT contents at 6-h intervals over 24-h and the excretion rates of urinary aMT6s at 3-h intervals over 24-h using RIAs. The pineal MT/urinary aMT6s relationship was assessed: (i) qualitatively, by comparing the circadian profiles of pineal MT content and urinary aMT6s excretion by 16-week-old gerbils; (ii) quantitatively, by correlating peak nocturnal pineal MT content with total urinary aMT6s pg/g BW/24-h.
d) To compare the rate and pattern of urinary aMT6s excreted by the BF and LF groups during sexual maturation.
e) To compare the circadian profiles of urinary aMT6s by the HF and LF groups at 11 Y2 weeks (sexual maturity) and at 16 weeks (adulthood) in order to discover whether F affects the rhythmicity of urinary aMT6s excretion, e.g., the amplitude, the time of appearance and decline of urinary aMT6s excretion, and the total amount of urinary aMT6s excreted during the daytime and night-time.
3. The purpose of the third experiment was to discover whether F affects the timing of the onset of sexual maturation in gerbils. The objective was to compare several physiological markers for the onset of puberty in the two groups, i.e., the areas of the ventral glands, age of vaginal opening, body weights and weights of testes.
4. The purpose of the fourth experiment was to demonstrate that F was the only variable between the two groups. The aim was to compare the [F] of gerbil bone ash from the HF and LF groups at various ages.
The project will provide basic information on the rate and circadian profiles of urinary aMT6s excretion during the development of the gerbil, a common species in pineal research. Such basic knowledge is a prerequisite for further studies using urinary aMT6s measurements as an alternative to pineal or plasma MT determinations in the gerbil. It was hoped that the results would contribute new knowledge on pineal MT output during puberty in gerbils and contribute towards knowledge about the pineal’s function during sexual development.
The results will add new knowledge about the fate and distribution of F in the human body. Although it is difficult to evaluate the relevance of gerbil data to the human situation, the results may suggest a relationship between F and the timing of the onset of human puberty. In this way, the work may help to evaluate the propriety of the current extensive use of F in dentistry, i.e., affirm its safety or intimate that F has physiopathological effects on the pineal gland.
Chapter 10 – Discussion
After half a century of the prophylactic use of fluorides in dentistry, we now know that fluoride readily accumulates in the human pineal gland. In fact, the aged pineal contains more fluoride than any other normal soft tissue. The concentration of fluoride in the pineal was significantly higher (p
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