The Effect of Fluoride on the Physiology of the Pineal Gland

THE EFFECT OF FLUORIDE PHYSIOLOGY

ON THE

OF THE PINEAL

GLAND

Jennifer Anne Luke

A dissertationsubmittedto the School of Biological Sciences, University of Surrey, in fulfilment of the requirements for the Degree of Doctor of Philosophy.

Guildford 1997

(ii)

ABSTRACT The purpose was to discover whether fluoride (F) accumulatesin the during thereby early pineal gland and affects pineal physiology development. The [F] of 11 aged human pineals and corresponding muscle were determined using the F-electrode following HN4DS/acid diffusion. The rnýan [F] of pineal was significantly higher (p < 0.001) than muscle: 296 ± 257 vs. 0.5 ± 0.4 mg/kg respectively. Secondly, a discover longituUinal to controlled experimental study was carried out during biosynthesis (MT), F the pubertal whether affects of melatonin, developmentusing the excretion rate of urinary 6-sulphatoxymelatonin, (aMT6s), as the index of pineal MT synthesis.Urine was collected at 3hourly intervals over 48 hours from two groups of gerbils, (Meriones (12 f, (IHF) 12 low-F high-F (LF) m/group): under unguiculatus), and LD: 12 12, from prepubescenceto reproductive maturity (at 9-12 weeks) to adulthood, i. e., at 7,9,11 V2and 16 weeks. The I-IF pups received 2.3 LF from days F/g BW/day birth BF 24 groups gg and whereafter until distilled food F/kg 37 7 received containing respectively and and mg The by levels Urinary water. aMT6s were measured radioimmunoassay. BF group excreted significantly less aMT6s than the LF group until the age of sexual maturation. At 11V2weeks,the circadian profile of aMT6s by the BF males was significantly diminished but, by 16 weeks, was inhibits MT F LF In to the pineal equivalent conclusion, males. synthesis in gerbils up until the time of sexual maturation. Finally, F development was associatedwith a significant acceleration of pubertal in female gerbils using body weights, age of vaginal opening and accelerateddevelopment of the ventral gland. At 16 weeks, the mean testesweight of BF males was significantly less (p < 0.002) than that of the LF males. The results suggest that F is associated with low leads levels MT to an accelerated sexual this circulating of and hypothesis in female The that the maturation gerbils. results strengthen the pineal has a role in pubertal development.

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DECLARATION 1, Jennifer Luke, hereby declare that this research paper is my own, unaided work. It is being submitted for the degree of Doctor of Philosophy in the University of Surrey, Guildford. It has not been presentedfor any degreeat any other University.

Iq

dayof

M"C"

1997

(iv)

In memory of Lynne 1943 - 1984

(v)

ACKNOWLEDGEMENTS It is a pleasure to thank everyone who helped me with this project. I husband, Dix, and help the acknowledgewith utmost appreciation of my from forbearance family: for interest, the their my encouragementand beginning, when the idea first dawned upon me, until now. Shaping the idea into the final form would certainly have been impossible without the help of colleaguesat the University of Surrey and without exception they were generousand patient in sharing their knowledge. In particular, I sincerely thank Professor Arendt for allowing the work to be carried logic. in I her laboratory impeccable for her gratefully of out sense and by Gordon Hartman, Dr the my acknowledge valuable assistancegiven been have doubt, this completed supervisor: without work would never English his Judy I to who, with patienceand without support. owe much humour, taught me the art of radioimmunoassaywork unfailing good for helped the urinary sulphatoxyand validate radioimmunoassay University Augusta, Whitford, Gary I Professor thank of melatonin. USA, for teaching me his method for fluoride analysis. I acknowledge Nicholas Porter for determining the levels of calcium in human pineal for his help Unit, in Animal Graham Moorey, the with the glands and Anatomy I the to the throughout twenty-four cycle. am grateful gerbils Department, UCL, for providing the human pineal glands. I am truly indebted to The Colt Foundation, The Heinz and Anna Kroch Foundation and The New Moorgate Trust Fund for their confidence in the work in the first place and for their generousfinancial assistance.

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LIST OF ABREVIATIONS 511T aMT6s ANOVA AUC B/Bo b.pt. BW CCT CNS CSF CSU Cv cpm DCC d-H20 F [F) GCMS h HA HF HIOMT HMDS L LD: 12 12 LF LFNM MT MW NAS NAT NSBs OH PC ppb

QCS

RIA rpm sc SCG SCGX SCN SD SEM SNK T/P ratio VS.

serotonin 6-sulphatoxymelatonin analysisof variance areaunderthe curve in RIA systems a commonresponsevariable boiling point body weight cranial computedtomogram centralnervoussystem fluid cerebrospinal. charcoalstrippedurine coefficient of variation countsper minute dextran-coatedcharcoal double-distilied water fluoride ion fluoride concentration spectrometry gaschromatography-mass hour hydroxyapatite high-fluoride group hydroxyindole-0-methyltransferase hexamethyldisiloxane Iitre 12hour light, 12hour dark cycle low-fluoride group low-fluoride non-monitoredgroup melatonin molecularweight N-acetylserotonin N-acetyltransferase non-specificbinding tubes hydroxylion pineal calcification part per billion quality control tubes radioimmunoassay revolutionsper minute subcutaneous superiorcervical ganglion superiorcervical ganglionectomy suprachiasmatic nuclei deviation standard standarderror of the mean Student-Newman-Keuls test tissuewater to plasmawater concentrationratio versus

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TABLE OF CONTENTS

M 00 Off) OV) (V) (VO (Vii) (X0 (Xvii)

Title Page Abstract Declaration Dedication Acknowledgments List qfAbbreviations Table qf Contents List qf Tables List qfFigures CHAPTER

I-

Literature

Review

I

1.1 Introduction

1

1.2 13

6

1.4 1.5 1.6 1.7 1.8 1.9 1.10

1.11 1.12 1.13 1.14

1.15 1.16

Review of the Literature Sourcesof Fluoride 1.3.1 Food 1.3.2 Drinking Water 1.3.3 Dental Products Fluoride in Plasma Fluoride in Calcified Tissues Fluoride in Soft Tissues Blood-Brain Barrier The Human Pineal Gland Pineal Physiology Factors Suggestingthat the Pineal Could Retain F 1.10 1 Pineal Calcification 1.10.2 Prevalenceof Pineal Calcification in Humans 1.10.3 Blood Supply Melatonin Synthesis Melatonin Metabolism of Melatonin Melatonin Production During Pubertal Development 1.14.1 Human Studies 1.14.2 Animal Studies 1.14.3 Sex Differences Measurementof Pineal Function Animal Model: the Mongolian Gerbil

8 10 11 12 14 17 18 19 20 23 23 28 31 31 35 35 36 37 39 40 40 43

(Viii) 1.17 Pineal Function in Gerbils 1.18 Age of SexualMaturity in Gerbils 1.18.1 Males 1.18.2 Females 1.19 Physiological Signs of SexualMaturation in Gerbils 1.19.1 Ventral Gland 1.19.2 Body Weights 1.19.3 Testes

45 46 46 46 47 47 49 49

CHAPTER

Aims and Objectives

51

Methodologies

54

2-

CHAPTER 33.1

3.2

3.3 3.4 3.5

Determination of the [F] of Pineal Gland, Bone and Muscle in Humans 3.1.1 Collection of the Samples 3.1.2 Analysis of Fluoride 3.1.2 (a) Separationof Fluoride 3.1.2 (b) Potentiometry 3.1.3 Experimental Procedurefor Analysis of F 3.1.3 (a) Apparatusand Reagents 3.1.3 (b) Methodology 3.1.3 (c) Limitations of HMDS Method 3.1.4 Modifications for Use with Various Tissues 3.1.4 (a) 3.1.4 (b) 3.1.4 (c) Determination of the [Ca] of Human Pineal Using Flame Atomic Absorption Spectrometry 3.2.1 Experimental Procedure 3.2.1 (a) Digestion of the Pineals 3.2.1 (b) Apparatus and Reagents 3.2.1 (c) Methodology Animal Maintenance Collection of Urine Samples Measurementof Urinary Sulphatoxymelatonin in Gerbils Using Radioimmunoassay 3.5.1 Principles of Radioimmunoassay 3.5.2 Materials 3.5.3 Validation of Assay for Use with Gerbil Urine 3.5.3 (a) Anti-serum dilution 3.5.3 (b) Cross-reactivity 3.5.3 (c) CSU on the standardcurve 3.5.3 (d) Parallelism 3.5.3 (e) Sensitivity 3.5.3 (f) Recoveries

54 55 57 59 63 65 66 67 68 68 69 69 69 70 71 73 75 75 76 80 81 82 83 83 84

(ix)

3.6

3.7 3.8

3.5.4 Methodology Measurementof Melatonin Content in Gerbil Pineal 3.6.1 Collection of the Gerbil Pineals 3.6.2 Analytical Procedure 3.6.3 Reagents 3.6.4 Methodology Physiological Signs of SexualMaturity in Gerbils Statistical Methods 3.8.1 The [F] of Human Tissues 3.8.2 Urinary aMT6s Levels as an Index of Pineal MT Synthesis 3.8.3 Urinary aMT6s Levels in Gerbils 3.8.4 The Circadian Rhythm of Urinary aMT6s in Gerbils at 11V2and16 Weeks 3.8.5 The Physiological Signs of Puberty 3.8.6 Description of the Experimental Groups

CHAPTER 44.1 4.2

Fluoride Contents of Human Tissues

Results Conclusions

84 87 88 89 91 92 93 93 93 94 95 96 96 97 97 99

CHAPTER 5-

Urinary Levels of aMT6s as an Index of Melatonin Synthesis in the Gerbil Pineal

104

5.1 5.2

104 108

Results Conclusions

CHAPTER 6-

Effects of Fluoride on Total

Urinary aMT6s Excretion by Gerbils 6.1 6.2 6.3

Results of the Longitudinal Study on Total aMT6s Excreted at 7,9,1 P/2and 16 Weeks Results of the Total aMT6s Excreted by All gerbils Aged 7 to 28 Weeks Conclusions

120 130

CHAPTER 7-

Effects of Fluoride on the Circadian Rhythm of Urinary aMT6s in Gerbils

7.1

Results of Circadian Profiles of Urinary aMT6s 7.1.1 At llV2Weeks 7.1.2 At 16 Weeks

135 135 135 143

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7.2 7.3

7.1.3 Comparisonof Profiles at 11V2and16 Weeks Intervals When the Gerbils Did Not Urinate Conclusions

146 149 150

CHAPTER 8- Effects of Fluoride on Physiological Signs of Puberty in Gerbils

158

8.1 8.2 8.3 8.4 8.5

158 159 160 161 162

Results of Areas of the Ventral Glands Results of Body Weights Results of Age at Vaginal Opening Results of TestesWeights Conclusions

CHAPTER 99.1 9.2

Fluoride Levels in Gerbil Bone

Results Conclusions

164 164 165

CHAPTER 10 - General Discussion and Conclusions

167

APPENDICES

178

A B C D E F

178 191 196 249 256 260

Resultsof F-Analyses Resultsof RIAs for PinealMT Contents Resultsof RIAs for aMT6sLevelsin Gerbil Urine DescriptiveStatisticsfor Urinary aMT6sLevels Resultsof the PhysiologicalSignsof Puberty Validation Results

REFERENCES

265

(xi)

LIST OF TABLES Table 1.1 Table 3.1 Table 3.2

[F] of bone ash in previous animal studies Age, gender,and causesof death of the subj ects

16 54

Cross-reactivity data for antiserum 1118/23884 Serial dilution of ten urine sampleswith CSU to show parallelism of urinary aMT6s values

81

Table 3.4

Quantitative recovery of unlabelled aMT6s

84

Table 4.1

The fluoride content of human pineal gland and bone (wet ash weight) and correspondingmuscle The calcium and hydroxyapatite content of human pineal glands Melatonin contentsin gerbil pineals at 16 weeks

104

Summary of total urinary aMT6s excretedby Y2and 16 7,9,11 weeks gerbils monitored at

113

Comparisonof the urinary aMT6s levels excreted by gerbils obtained from Section 6.1 and 6.2

121

Table 3.3

Table 4.2 Table 5.1 Table 6.1 Table 6.2 Table 6.3 Table 7.1 Table 7.2 Table 7.3 Table 8.1 Table 8.2 Table 8.3 Table 8.4

83

Summaryof total urinary aMT6s excretedby 28 Y2,16 7,9,11 weeks and all gerbils at Summary of the mean 48-h profiles of aMT6s in gerbils aged II Y2weeks

97 98

123 136

Summary of the mean circadian profiles of in aMT6s gerbils aged 16 weeks

144

Intervals when the gerbils did not urinate

150

Ventral gland formation in female gerbils at II from the LF and HF groups weeks

V2

158 159

Body weights (g) of gerbils aged 7 to 28 weeks Comparison of the number of females from HF and LF showing vaginal introitus at 7 weeks

161

Testesweights of gerbils at 9,16 and 28 weeks

162

(xii) Table 9.1

Fluoride content of gerbil bone ash

164

Results of the [F] of agedhumanpineal glands Results of the [F] of human bone ash

178

Results of the [F] of human muscle Results of the [Ca] of human pineals

181

Appendices Table A. I Table A. 2 Table A. 3 Table A. 4 Table A. 5 Table A. 6 Table A. 7 Table A. 8 Table A. 9

Results of the [F] of bone ash from LF males at 10 weeks and LF femalesat 11 weeks Results of the [F] of bone ash from HF males at 10 weeks and HF femalesat 11 weeks Results of the [F] of bone ash from HF males females HF and given HF diet after weaning

Table B. 2 Table B.3 Table BA Table B. 5

Table C. I Table C.2

182 183 184 185

Results of the [F] of bone ash from LF gerbils (male and female mixed) at 16 weeks

186

Results of the [F] of bone ash from FIF male and HF female gerbils aged 16 weeks

187

Table A. 10 Results of the [F] of bone ash from LF male female LF and gerbils aged28 weeks Table A. 11 Results of the [F] of bone ash from HF male and HF female gerbils aged28 weeks Table B. I

179

188 190

Results of the intra-assayand inter-assay coefficients of variation for pineal melatonin

191

Results of RIA for pineal melatonin contents in LF gerbils at 16-weeks(LFNM group)

192

Results of RIA for pineal melatonin contents in LF gerbils at 16-weeks

193

Inter-assayvariability of RIM for aMT6s by 16BF, 16LF, and 16LF NM groups

194

Results of pineal melatonin contentsat 0400 and corresponding24-h urinary aMT6s levels

195

Results of intra-assaycoefficients of for urinary aMT6s -variation

196

The inter-assaycoefficients of variation for RIAs for aMT6s by gerbils in Section 6.1

197

(Xiii) Table C.3 Table CA Table C.5

The inter-assaycoefficients of variation for all RIAs for urinary aMT6s The inter-assaycoefficients of variation for RIAs for aMT6s by gerbils at 11V2and16 weeks The CV% betweenlevels of aMT6s in urine in day day by 7 I 2 collected gerbils at weeks and

The CV% betweenlevels of aMT6s in urine in day day by I 2 collected and gerbils at 9 weeks Table C.7 The CV% betweenlevels of aMT6s in urine in by II V2 day day 2 I weeks collected and gerbils at Table C.8 The CV% betweenthe urinary aMT6s levels in in Section 6.2 day day by 2 1 collected and gerbils Table C.9 The CV% betweenthe urinary aMT6s levels in 6.2 Section in day day by 2 I collected gerbils and Table C. 10 The CV% betweenurinary aMT6s levels day by 28 in day 2 I at weeks gerbils collected and

198 199 200

Table C.6

201 202 203 204 205

Table C. 11 Results of the longitudinal study of urinary aMT6s by female gerbils at 7,9,11 1/2and16 weeks

206

Table C. 12 Results of the longitudinal study of urinary aMT6s by male gerbils at 7,9,11 V2and16 weeks

207

Table C. 13 Laboratory results of urinary aMT6s 7 female by LF gerbils at weeks excreted

208

Table C. 14 Laboratory results of urinary aMT6s female 7 by HIF gerbils at weeks excreted

209

Table C. 15 Laboratory results of urinary aMT6s 7 by LF at weeks excreted male gerbils

210

Table C. 16 Laboratory results of urinary aMT6s 7 by BF excreted male gerbils at weeks

211

Table C. 17 Laboratory results of urinary aMT6s female by 9 LF excreted gerbils at weeks

212

Table C. 18 Laboratory results of urinary aMT6s female 9 by BF excreted gerbils at weeks

213

Table C. 19 Laboratory results of urinary aMT6s by 9 LF excreted male gerbils at weeks Table C.20 Laboratory results of urinary aMT6s excretedby HF male gerbils at 9 weeks

214 215

(Xiv) Table C.21 Laboratory results of urinary aMT6s excretedby LF female gerbils at 11V2weeks Table C.22 Laboratory results of urinary aMT6s by female HF excreted gerbils at 11V2weeks Table C.23 Laboratory results of urinary aMT6s by V2weeks LF 11 excreted male gerbils at Table C.24 Laboratory results of urinary aMT6s excretedby HF male gerbils at II Y2weeks Table C.25 Laboratory results of urinary aMT6s by female LF excreted gerbils at 16 weeks Table C.26 Laboratory results of urinary aMT6s by female HF excreted gerbils at 16 weeks Table C.27 Laboratory results of urinary aMT6s excretedby LF male gerbils at 16 weeks Table C.28 Laboratory results of urinary aMT6s excretedby HF male gerbils at 16 weeks Table C.29 Laboratory results of urinary aMT6s by female gerbils at 11Y2weeksin 2x 24-h collections Table C.30 Laboratory results of urinary aMT6s by in 24-h 2x collections pubertal male gerbils Table C.31 Laboratory results of urinary aMT6s by in 24-h 16 2x collections gerbils at weeks Table C.32 Laboratory results of urinary aMT6s by female gerbils at 28 weeks in 2x 24-h collections Table C.33 Laboratory results of urinary aMT6s by in 2x 24-h 28 collections male gerbils at weeks

216 219 222

225 228 229 230 231 232 233 234 235 236

Table C.34 Circadian excretion of urinary aMT6s (ng/3-h) by LF female gerbils aged II Y2weeks

237

Table C.35 Circadian excretion of urinary aMT6s (ng/3-h) by IF female gerbils aged II V2weeks

238

Table C.36 Circadian excretion of urinary aMT6s (ng/3-h) by LF male gerbils aged II Y2weeks Table C.37 Circadian excretion of urinary aMT6s (ng/3-h) by BF male gerbils aged 11Y2weeks, Table C.38 Circadian excretion of urinary aMT6s (pg/g BW/3-h) by LF femalesat 11Y2weeks

239 240 241

(XV) Table C.39 Circadian excretion of urinary aMT6s (pg/g BW/3-h) by I-IF femalesat 11V2weeks

242

Table C.40 Circadian excretion of urinary aMT6s (pg/g BW/3-h) by LF males at 11V2weeks

243

Table C.41 Circadian excretion of urinary aMT6s (pg/g BW/3-h) by HF males at 111/2weeks

244

Table C.42 Circadian excretion of urinary aMT6s (ng/3-h) by LF female gerbils aged 16 weeks Table C.43 Circadian excretion of urinary aMT6s (pg/g BW/3-h) by LF femalesat 16 weeks Table C.44 Circadian excretion of urinary aMT6s (ng/3-h) by IHF female gerbils at 16 weeks Table C.45 Circadian excretion of urinary aMT6s (pg/g BW/3-h) by BF femalesat 16 weeks Table C.46 Circadian excretion of urinary aMT6s (ng/3-h) by LF male gerbils aged 16 weeks Table C.47 Circadian excretion of urinary aMT6s (pg/g BW/3-h) by LF males at 16 weeks Table C.48 Circadian excretion of urinary aMT6s (ng/3-h) by HF male gerbils aged 16 weeks Table C.49 Circadian excretion of urinary aMT6s (pg/g BW/3-h) by HIFmales at 16 weeks Table D. I Table D. 2 Table D. 3 Table DA Table D. 5 Table D. 6 Table D. 7

Descriptive statistics for the daily urinary aMT6s levels excretedby gerbils aged 7 weeks Descriptive statistics for the daily urinary aMT6s levels excretedby gerbils aged9 weeks Descriptive statistics for the daily urinary aMT6s levels excretedby gerbils aged 11V2weeks, Descriptive statistics for the daily urinary aMT6s levels excretedby gerbils aged 16 weeks Descriptive statistics for the daily urinary aMT6s levels excretedby all male gerbils aged 9 weeks

245 245 246 246 247 247 248 248

249 250 251 252 253

Descriptive statistics for the daily urinary aMT6s levels excretedby all gerbils aged 11V2weeks

254

Descriptive statistics for the daily urinary aMT6s levels excreted by all gerbils aged 16 weeks

255

(Xvi) Table D. 8

Table E. I Table E.2 Table E.3 Table EA Table F. 1 Table F.2

Descriptive statistics for the daily urinary aMT6s levels excretedby gerbils at 28 weeks

256

The areasof the ventral glands in male female 10 28 and and weeks gerbils at -

257

t- tests betweenthe body weights of gerbils from the BF and LF groups at 7,9 and 111/2weeks t- tests betweenthe body weights of gerbils from the HF and LF groups at 16 and 28 weeks The combined testesweights of gerbils at 16 weeks Results of antiserumdilution curves Limit of detection of RIAs for urinary aMT6s

258 259 260 261 262

(Xvii)

LIST OF FIGURES Fig. 1.1 PlasmaF-concentrationsin subjectsliving in a 1.2 mg F/L areaand 0.25 mg F/L area Fig. 1.2 PlasmaF-concentrationsafter intake of four different oral dosesof F Fig. 1.3 Indole metabolism in the mammalianpineal gland

12 14 33

Fig. 1.4 A family of Mongolian gerbils (Meriones unguiculatus) Fig. 1.5 The superficial pineal gland of the gerbil at 12 weeks

43

Fig. 1.6 The ventral gland in the gerbil Fig. 3.1 Typical standardcurve for F ion-specific electrode

48

Fig. 3.2 Fig. 3.3 Fig. 3.4 Fig. 3.5 Fig. 5.1 Fig. 5.2 Fig. 6.1 Fig. 6.2 Fig. 6.3 Fig. 6.4 Fig. 6.5

Diagram to show main featuresof HMDS-facilitated F for isolation ionic method and acid-ionizable of Diagram of metaboleused to collect the gerbil urine Antiserum dilution curves for aMT6s The effects of CSU on the standardcurve for aMT6s Circadian profiles of aMT6s in gerbils at 16 weeks Circadian profile of MT in gerbil pineals and aMT6s excretion rates (pg/g BW/3-h) at 16 weeks Comparison of levels of aMT6s (ng/24-h) excreted by BF and LF groups at 7,9,11 V2and16 weeks Comparison of levels of aMT6s (ng/24-h) excreted by the sexesat 7,9,11 V2and16 weeks Comparison of relative levels of aMT6s excreted by IHFand LF groups at 7,9,11 V2and16 weeks Comparison of relative levels of aMT6s,excreted by the sexesat 7,9,11 V2and16 weeks Comparison of levels of aMT6s (ng/24-h) excreted V2,16 by IHFand LF groups at 7,9,11 28 and weeks

44 58 63 74 80 82 106 107 114 116 117 119 124

(Xviii) Fig. 6.6 Fig. 6.7 Fig. 6.8 Fig. 7.1 Fig. 7.2 Fig. 7.3 Fig. 7.4 Fig. 7.5 Fig. 7.6

Comparisonof levels of aMT6s (ng/24-h) excreted V2,16 by the sexesat 7,9,11 and 28 weeks Comparison of relative levels of aMT6s excreted by BF and LF groups at 7,9,111/2,16 and 28 weeks Comparison of relative levels of aMT6s excreted V2,16 by the sexesat 7,9,11 28 and weeks Comparisonof the absolute48-h profiles of aMT6s in gerbils from the BF and LF groups at 11V2weeks Comparisonof the relative 48-h profiles of aMT6s in gerbils from the HF and LF groups at 11V2weeks Comparison of the absoluteand relative 48-h profiles females V2weeks in 11 LF LF at of aMT6s males and Comparison of the absoluteand relative 48-h profiles of aMT6s in I-IF males and BF femalesat 11V2weeks, Comparison of relative 48-h profiles of aMT6s in LF males, LF females and I-IF femalesat II 1/2weeks Comparison of circadian profiles of aMT6s in interval during 3-h gerbils which urinated every

Fig. 7.7 The absolute circadian profiles of aMT6s in gerbils aged 16 weeks from the HIFand LF groups Fig. 7.8 The relative circadian profiles of aMT6s in from 16 LF HIF the at weeks gerbils and groups Fig. 7.9 Comparisonof the absolute and relative profiles of aMT6s in LF gerbils at 11V2and-16 weeks Fig. 7.10 Comparison of the absolute and relative profiles in 16 females V2and HF II weeks at of aMT6s Fig. 7.11 Comparison of the absolute and relative profiles in 16 II 1/2and HF weeks of aMT6s males at Fig. 8.1 Body weights (g) of gerbils from BF and LF from 7 to 28 weeks of age groups Fig. 8.2 Cumulative percentageof age at vaginal opening in females from HF and LF groups

126 128 130 137 139 140 141 142 143 145 146 147 148 149 160 160

I

Chapter 1- Background Information 1.1 Introduction

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 elsewherein the body. It is remarkable that the pineal gland has never been analysed separatelyfor F becauseit has several featureswhich suggestthat it highest has F. It the could accumulate calcium concentration of any in in it body because tissue the normal soft calcifies physiologically the form of hydroxyapatite (HA). It has a high metabolic activity blood factors favouring two coupled with a very profuse supply: the deposition of F in mineralizing tissues. The fact that the pineal is outside the blood-brain barrier suggests that pineal HA could from F the bloodstream if it has the same strong affinity sequester for F as HA in the other mineralizing tissues.

2

The intensity of the toxic effects of most drugs dependsupon their concentration at the site of action. The mineralizing tissues (bone first high F teeth) the to and accumulate concentrationsof and are show toxic reactions to F. Hence, their reactions to F have been in If F especially well studied. accumulates 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. Children are now exposedto more F than ever before. Fluorides are the cornerstoneof 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 accessto fluoridated water supplies and the increasedexposureto F in dental important is fluoridation The public products. of water supplies an 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 F344/N rats which had received drinking water containing 100-175 mg NaFAL (45-79 mg F/L) for two years (NTP, 1990). Following this report, three critical bodies analysedthe public health benefits and risks from chronic Fexposure by reviewing the evidence from human epidemiological studies of the relationship between cancer and water fluoridation and also carcinogenicity studies in rodents. They unanimously

3

is F that agreed safe and effective if used appropriately. The use of F is not associatedwith an increasedcancerrisk 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 researchis required on the effects of F on the reproductive systemin animals and humans(USPHS, 1991). Dental fluorosis (defective, hypomineralized enamel) occurs when its during F tooth the excessive amounts of reach growing 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 body F/kg 0.1 daily intake mg population correspondsto a of about firm is (13W) 12 there to the no weight years although up age of issue. fact, high In this consensuson prevalence and severity of a dental fluorosis was reported in populations with an estimateddaily F-intake of less than 0.03 mg F/kg BW (Bwlum et al, 1987). The so-called 'optimal' concentration of F in community water is defined as the concentration of F which gives maximum caries fluorosis, i. between 0.7 dental e., reduction and causes minimum depending At 1.2 the temperature. the and mg/L on mean ambient time of Dean's original studies, there was a 10-12 percent prevalence of mild dental fluorosis in children in the I mg F/L (Dean for in 1941,1942). 'accepted' This the areas et al, return was benefits in caries reduction: a classic public health trade-off. In the 1930sand 1940s,virtually the only source of F was in the drinking

4

is F Today, in water. ubiquitous the environment which meansthat daily F-intake from besides tap water. man's comes severalsources Systemic F-exposure to children has increased (Leverett, 1991). Mild dental fluorosis is now more common than one would predict basis in findings in late Dearf 1940s: 1930s the the on of s and early fluoridated and non-fluoridated communities (Leverett, 1986; Pendrys and Stamm, 1990; USPHS, 1991). Several recent studies report prevalencerates in the 20 and 80 percent range in areaswith fluoridated water (Levy, 1994). The prevalence of 0.9 percent (recorded in the pre-fluoride days) in areascontaining less than 0.4 in (USPHS, has increased 1991). FAI, 6.6 to the mg water percent The prevalenceof moderateto severedental fluorosis has increased (Williams and Zwemer, 1990; USPHS, 1991; Lalumandier and Rozier, 1995). The increased prevalence of dental fluorosis is it is because the an causing concern within scientific community early sign of F-toxicity and evidence that some children are now issue has is fo The F the potential to them. than getting more good r becomea signiflcant dental health problem. Of all the tissues, the developing enamel organ is assumedto be F. It to the toxic of contains significantly most sensitive effects higher concentrations of F and calcium than other soft tissues. The higher F9-day-old enamel organs of rats contained significantly 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 reachedpeak values (0.19 mg/kg) in 30 increased The be to minutes. enamel organ may relatively sensitive F-intake because 1992). it (Bawden F systemic et al, accumulates

5

Although the exact mechanismresponsible for enamel fluorosis is known, have F may not specific effects on the normal activity of in developing ameloblasts, enamel matrix and proteolytic, activity the maturing enamel (DenBesten. and Thariani, 1992). The transition/early-maturation stage of

is amelogenesis

most

F-concentration. increased to the susceptible effects of an plasma The aesthetically important maxillary central incisors are most Stamm, 1991). (Evans F 22-26 to vulnerable at months and Alongside the calcification in the developing enamel organ, is in It is the a normal child's pineal. calcification also occurring A complex series of enzymatic reactions physiological process. the pinealocytes converts the essential amino acid, tryptophan, to a whole family of indoles. The main pineal hormone

within

is melatonin (MT). For some reason, young children have the highest levels of plasma MT. They also have higher plasma Flevels (recommendedfrom a dental perspective) than they did 50 from increasing An mild number of children suffer years ago. dental fluorosis: evidence that they received too much F during the first few years of life. If F accumulatesin the pineal gland during indole In it metabolism. much early childhood, could affect pineal the same way that high local concentrations of F in enamel organ bone and affect the metabolism of ameloblastsand 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 involvementin the onsetof puberty.If F compromisespineal function by altering

6

the high rate of synthesis of MT during childhood, does this mani est 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 suggestthat harmful effects on human health have already occurred: either way, this investigation is worthwhile. 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 demonstratethe safety of water fluoridation. The New York StateDepartment of Health initiated the study in 1944 becausethey realized that there would ultimately be a need for a long-term evaluation of any possible systemic effects as well as the dental changesfrom drinking fluoridated water over a long period of time.

Similar groupsof childrenwere selectedfor long-termobservation from Newburgh (fluoridated to 1.0 to 1.2 mg4L in 1945) and Kingston (essentially F-free for the duration of the study). Newburgh and Kingston were chosen becausethey were wellmatched:both were situatedon the HudsonRiver 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

7

birth from Newburgh, (aged In 817 to nine care,etc. children out of in 1954in 1945,500 years) who were selected were examined 1955; in Kingston, out of 711 children who were selectedin 1945, 405 were examined in 1954-1955. The medical and dental examinations began in 1944, and were An 1955. repeated periodically until assessmentof any possible fluoridated from the water systemic effects arising consumption of 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 index in of the rate of sexual menstruation girls was used as an maturation.

At the end of ten years, the investigatorsreported no adverse because fluoridated from drinking no water systemic effects from found differences between the two the results significant were first The groups. averageageof menarchewas earlier amonggirls in Newburghthan those in Kingston: 12 yearsvs. 12 yearsand 5 1956). Although (Schlesinger this months respectively et al, difference was not considered important, it does suggest an drinking fluoridated between the water and an use of association had The Newburgh in girls earlier onsetof sexualmaturation girls. first For fluoridated had lifelong the two years water. use of not a or so, they received unfluoridatedwater. Furthermore,their only from drinking F the sourceof was water.

8

1.3 Sources of Fluoride 1.3.1 Food

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): regardlessof the F-intake to the nursing mother. Ekstrand and co-workers (1981) analysedplasma and milk samplesfrom five nursing mothers after they had taken an oral dose of 1.5 mg F. There was an immediate ten-fold increasein the [F] of plasma (70-86 ng/ml) within 30 minutes of dosing but the [F] of breast day (2-8 throughout the milk remained constant ng/ml). The mean F-concentrations of human breast milk were 8.9 and 5.0 from living in 1.7 and 0.2 mg FALareas ng/ml nursing mothers 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 F/L areasrespectively (Spak et al, 1983). The reason for the limited transfer of F from plasma to breast milk is unknown. It has been suggestedthat the physiological barrier from the the newborn plasma-milk actively protects 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) in are 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

9

into fluids the and subsequentlyexcreted released extracellular into urine. Therefore, early human development has always in high-F in F-free the areas:a milieu even occurred a virtually lasts until the age of weaning and the phenomenon which introduction of solid foods. In contrast, the F-intake to bottle-fed infants living

in

fluoridated areasdependsupon the [F] of: a) the water used to itself formula-feed feed; b) the the reconstitute powdered 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 gg/day (Ekstrand, 1989). The normal is during infancy Bottle-fed F reversed. pharmacokinetics of 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 fruits, vegetables, pulses, roots, nuts, etc., rarely contain more than 0.2 to 0.3 mg F/kg (WHO, 1984). Most plant specieshave Ffrom limited F the to soil even when absorb a capacity flesh (Davison, The fertilisers 1984). of containing are applied bone), low levels from fish, (free of contains meat, poultry and F becausevirtually all F in animals occurs in their bones arid teeth. The skin and bones of tinned salmon and sardines fish because F/kg 8 500 the are contain and mg respectively in (1.2-1.4 high F levels to seamg/L) exposed relatively of (Jenkins, 1990). water

10

Nevertheless, most of man's daily intake of F is derived from food unless he drinks fluoridated water (or water containing naturally high levels of F) or is exposed to F from industrial contamination (Underwood, 1977). Studies have shown that adults living in non-fluoridated areas receive low levels of F from food alone: 0.16 mg F/day (Machle et al, 1942); 0.2 to 0.3 mg F/day (McClure, 1949); 15- to 19-year-old American males 0.27 ± 0.03 mg F/day (Singer et al, 1985). Six-monthreceive old infants and two-year-old children receive less than 0.2 mg F/day from food (Ophaug et al, 1980a; 1980b). The dispersion of F into food was an unplanned consequence of water fluoridation. Processedfood and beverages contain significant amounts of F if they are manufactured in towns be fluoridated foodstuffs Such supplied with water. can distributed and consumed in non-fluoridated communities. Their consumption produces an averageF-intake of 1.0 to 2.0 mg/day (Marier and Rose, 1966). 1.3.2 Drinking Water

Man has evolved over the millenia drinking water with low Flevels, e.g., surface waters, collected rain or melted snow. Today, most large communitiesobtain their drinking water from suppliesof fresh surfacewaters which contain between 0.01 to 0.3 mg F/L (WHO, 1984).In the 1940s,95.7% of the populationof USA receivedwater containinglessthan 0.5 mg FAL(Hill et al, 1949);in the 1980ý,most water suppliesin the USA contained less than 0.3 mg FAL unless they had been

II

artificially fluoridated (Centers for Disease Control, 1985); in Norway, 96% of the population consumed water containing less than 0.25 mg F/L (L6kken and Birkeland, 1978). In someparts of the world, particularly rural areas,populations use water from deep wells or bores. Underground water has a greater opportunity to contact fluoriferous material. Groundwaters contain varying amounts of F: less than 0.1 mg/L to more than 25 mg/L (USPHS, 1991). Bottled spring waters (still) contain less than 0.1 mg FAL. The drinking water from wells in districts with endemic fluorosis in USA contained 2.0 to 16.8 mg FAL(McKay, 1933).

1.3.3 Dental Products

Since 1945,therehasbeena steadyincreasein the useof F in forms for dental the various and concentrations preventionof caries.The self-appliedtopical fluorides include: fluoridatedtoothpaste(1000-1500mg F/kg) which typically provides0.51.5 mg F per brushing when 0.5-1.5 g toothpaste(1000 mg F/kg) is dispensed;cosmeticmouth-washsold over the counter (230 mg FAL)provides 1.1-3.4mg F when 5-15 ml is used; high-F mouth-rinsesby prescription(900 mg FAL)(Shulmanet 1995). The al, professionally-appliedtopical fluorides include: F-gels (12,300 mg F/kg); F-varnishes. The F-preparations intendedfor systemicuseincludeF-tabletsandF-drops. Young children swallow substantial amounts of toothpaste or mouth-rinse because they have poor control over the

12

is in The F toothpaste swallowing reflex. and mouth-rinse bioavailable 1980) (Ekstrand Ehmebo, and readily and is in ingested F the virtually all absorbed the gastrointestinal five60% A two-year-old tract. and a child swallows about year-old about 34% of the F in toothpastes(Simard et al, 1989, 1991). In real terms, 2-5-year-old children ingest 0.5 to 0.75 ingest 0.4 F (Murray, 1986); 7-13-year-old children mg per use to 1.2 mg F per use (Bell et al, 1985). Therefore, some children from daily F-allowance their toothpaste alone. exceed optimal Consequently,there is an increasein plasma F-levels.

1.4 Fluoride in Plasma

In low-F areas,the mean [F] in plasma from fasting, middle-aged 0.5 is from 0.004 0.02 0.01 to mg/L or adults about mg/L ranging F-levels fluoridated (Murray 1991). In the areas, plasma gM et al, (Ekstrand 1988). from I 0.02 0.04 to et al, range mg/L, about gM

60

40

20

0

8a. m.

8 p.m.

8 a.m.

8P.m.

Fig 1.1 Plasma F-concentrations in five subjects living in a 1.2 mg F/L area since birth (A ) and the mean plasma F-concentration from five subjects living in a 0.25 mg FAL area since birth (o Reproducedfrom Ekstrand, 1978, with permission.

13

Figure 1.1 presentsthe plasma [F] curves from five subjects from areaswith 1.2 and 0.25 mg F/L in the water supply. To simplify the from from five [F] 0.25 the the the graph, mg mean plasma subjects FAL area is presented because their plasma [F] were similar throughout the 24-h period (9.4 ± 2.2 ng/ml, ± SD). The plasma [F] in subjects from the fluoridated area were higher than those in the low-F subjects throughout the day and two subjects in the high-F diurnal (Ekstrand, 1978). areashowedsigns of a variation Less is known about the [F] in plasma from infants and young is in [F] The the children. plasma suckling rat very low: about 0.002 mg/L (Bawden et al, 1986; 1992) which is probably due to the low [F] of rat milk (less than 0.01 mg/L) (Drinkard et al, 1985). Presumably the plasma [F] in infants are also very low: only increasingwhen F is administered.Fluoride is rapidly cleared from the bloodstream in infants and young children by sequestrationby the rapidly mineralizing tissues. Figure 1.2 presents the 24-h plasma [F] in a male subject in four four in he different single separateexperiments which was given be is doses (Ekstrand F F It 1977). that of can seen oral et al, Ffrom The the tract. rapidly and readily absorbed gastrointestinal levels in plasma start to rise within a few minutes after F-intake and take 30 minutes to reach the peak values and severalhours to return to basal levels.

Plasma F-levels are not homeostatically regulated in the classical Peak ingested F the to sense. values are proportional and amount of I

body 1988). (Ekstrand to proportional et al, inversely weight

14 Plasma fluoride, pM 251 10

5.0 2.5 1.0 0.5 0.25 12 Hours

Fig. 1.2 Plasma F-concentrations in a healthy male aged 23 years intake four different after an of oral doses of F. Reproduced from Ekstrand et al, 1977, with permission. Young children regularly receive repeated doses of F from swallowing fluoridated toothpaste. When 3- to 4-year-old children ingested 0.6 g toothpaste(1000 mg F/kg), the mean peak plasma F(4 concentration [M) was almost the same as when they received 0.5 mg NaF tablet (Ekstrand et al, 1983). One hour after the routine application of topical F-gels to the teeth, children had peak plasma F-levels ranging from 16 to 76 pM. The highest value (76 pM F) in The F-levels 5-year-old high occurred a child. plasma remained for two hours (Ekstrand et al, 1981).

1.5 Fluoride in Calcified Tissues

Approximately 99% of all the F in the human body is found is because F tissues associatedwith calcified avidly attractedto hydroxyapatite(HA). The rapid uptake of F by the mineralized tissuesis one reasonfor the low levels of F in plasma(Jenkins,

15

1990). The rate of clearance of F from plasma exceeds that for calcium (Costeas et al, 1971). Whole enamel contains about 100 F/kg mg although the outer 2 gm of surface enamel from permanent teeth contains high levels of F- 1000-3000 mg/kg dependingupon the [F] of the drinking water (Murray et al, 1991). Dentine and cementurn contain about 300 and 1000 mg F/kg respectively (Newbrun, 1986). Bone is the main store of F and its [F] is a good indicator of previous F-exposure. Bone F-content directly correlates with Fintake (Turner et al, 1992); with age of the subject, and the duration of exposure (Kuo and Stamm, 1974; Parkins et al, 1974; Charen et al, 1979). Fluoride is not evenly distributed throughout bone. The [F] of trabecular bone is higher than cortical bone. The [F] of endostealand periosteal surfacesare higher than the central bone. deposition The of parts of F also dependsupon the stage of development, rate of growth and the vascularity of the bone; the surface area and size of the crystallites. Therefore, when bone samples are taken for F-analysis, the sites must be standardized (Jenkins, 1990).

Under normal circumstances,young or middle-agedadults retain half aboutone of the absorbedF in the calcified tissuesand excrete the otherhalf in the urine. Infantsandyoung childrenretain moreF than adults because they have a greater proportion of their skeletonsavailableto the circulation and are actively laying down bonemineral (Whitford, 1996).Infants and young children excrete lessof the daily F-intakein the urine.

16

Several studies have determined the [F] of human bone. The mean [F] of bone ash from elderly subjects was: 2085 ± 1113 mg/kg (Charen et al, 1979); 1834 mg/kg and the highest value was in a 79-year-old subject: 3708 mg F/kg (Ebie et al, 1992). Jenkins (1990) reported F-levels as high as 6000 mg/kg in bone ash from elderly subjects living

in Hartlepool where water supplies

contained 2 mg FALfor most of the past 100 years. Table 1.1 presentsthe F-concentrations of ashedbone in previous laboratory studies in which the animals were given different daily dosesof F in either the drinking water or the food. Table 1.1 Fluoride content of bone ash in previous animal studies bone[F] food water Reference Species Duration mg F/L

rat rat rat rat rat rat rat mouse rat mouse rat

15 15 50 50 -30 75 45 tap tap tap tap

mg F/kg

12w 24 w 12w 24 w 6w 6w 103 w -8 4 mg NaF/kg BW/d 95 w 4 mg NaF/kg BW/d 95 w 10 mg NaF/kg BW/d 95 w 10 mg NaF/kg BW/d 95 w 20 000 > 20 000

5-methoxyindole aceticacid

> 20 000

> 0.11

5-hydroxyindole acetic acid N-Acetyltryptamine N-Acetylserotonin N- Acetyltryptophan 5-hydroxytryptophan N-methyltryptamine 5-methoxytryptamine 5-methoxytryptophan 5-hydroxytryptarnine 5-methoxytryptaphol 5-hydroxytryptaphol Tryptophan

> 20 000 > 20 000 > 20 000 > 20 000 > 20 000 > 20 000 > 20 000 > 20 000 > 20 000 > 20 000 > 20 000 >2 000 000

> 0.11 > 0.11 > 0.11 > 0.11 > 0.11 > 0.11 > 0.11 > 0.11 > 0.11 > 0.11 > 0.11 > 1.1 x 10-1

of

aMT6s 5-sulphatoxy-N-acetylserotonin 5-glucuronide-N-acetylserotonin 6-glucuronide melatonin 6-hydroxymelatonin

T3 -

from (1988) Aldous Arendt and Reproduced

100.0 2.0 1.4 0.5 > 0.11 > 0.11

82

The specificity of the anti-aMT6s antiserum, (Batch No 1118/23884), was assessedby comparing the ability of several indoles and indole metabolites to displace 50% of maximum 125I-aMT6s antibody-bound under the conditions routinely employed in the assay. Table 3.2 shows that the percentagecross-reactivity with all the tested compounds was negligible and so they would not compete with aMT6s in the assay. Only 5-sulphatoxy-Nacetylserotonin,

5-glucuronide-N-acetylserotonin and

6-

glucuronide melatonin had low cross-reactivities but as they are present in such small amounts in urine, the antiserum was considered to be sufficiently specific for clinical application without pre-assaytreatment.

3.5.3 (c) Effect of CSU on the standard curve

100-

+1-2gl CSU CSU ýtl -v-5 CSU 10 111 -c-CSU, ul -*-25

so604020 0--

0.1

1

10

100

massaMT6s (ng/ml)

Fig. 3.5 The effects of increasing amounts of gerbil charcoal stripped urine on the standardcurve for urinary aMT6s

83

The addition of increasing volumes of gerbil CSU (2-25 pl) had no effect on the standardcurve for aMT6s.

3.5.3 (d) Parallelism

To determine whether the assay was parallel, ten samples of in CSU 4 diluted 1: 2 1: and gerbil and gerbil urine were diluted (Table The 3.3) that the samples show assayed. results parallel. Table 3.3 Serial dilution of ten urine samples with CSU to show parallelism of urinary aMT6s values (ng/ml) urinary aMT6s Sample

initial

1: 2 CSU

1: 4 CSU

30.6 27.3 17.1 10.3 8.2 20.6 5.5 12.5 15.3 15.8

16.2 13.1 8.6 6.1 4.1 11.5 2.7 5.2 7.6 9.2

concentration 1 2 3 4 5 6 7 8 9 10

55.4 50.6 32.5 21.5 15.4 42.8 11.5 22.6 32.7 37.6

3.5.3 (e) Sensitivity

The sensitivity of the assay, defined as the least amount distinguishablefrom zero at the 95% confidencelevel, was 0.2 in Appendix. See Tables F. 2(i)-F. 2(iii) ng aMT6s/tube.

84

3.5.3 (f) Recoveries

Table 3.4 showsthat the analyticalrecoveryof aMT6s at three different concentrationswasbetween98.3and 102.8%(n = 10). Table 3.4 Quantitative recovery of unlabelled aMT6s sample 0 0.5 1

15 ng/ml aMT6s 30 ng/ml aMT6s 5 ng/ml aMT6s added added added -FO/o FI/o total recovered total recovered total % recovered 5.8 6.5 6.2

106 114 94

7.2

112 93 97 95

31.6 32.8 30.5

104 107 97

96

17.3 14.8 16.0 16.7

32.4

100

2 3 4

0.8 1.5 2.4

5

0.4

5.9

110

15.4

100

29.3

96

6

1.7

6.5

96

16.5

99

31.5

99

7 8

4.6 3.7

9.3

94

18.3

91

33.9

98

102

9

1.2

8.8 6.5

106

17.6 16.7

93 103

33.0 31.9

98 102

10

1 7.2 1

12.7

110

22.2

100

34.6

91

mean recovery SD

102.8 7.4

98.3 6.1

99.2 4.3

Sections 3.5.3 (c), (d) and (f) were kindly performed by Judy English.

3.5.4 Methodology

The assayprocedurewasbasedon that previouslydescribedby Aldous and Arendt (1988) with the following modifications. 200 ýLlof antiserumwas used at a dilution of 1:40 000 in buffer. Gerbil CSUwasusedinsteadof humanCSU. Dilution of the samplesand QCs: The urine samples(which had been diluted with d-H20 following rinsing of the metaboles) were further diluted with tricine buffer using an automatic diluter. The degree of dilution depended upon the

original dilution of the urine collected from the metaboles.

85

Urine samplescollected at 3-h and 24-h intervals were diluted 1:25 and 1:50 respectively. QCs were diluted 1:25. The levels of urinary aMT6s excreted by the 7- and 9-week-old gerbils were assayedon a 24-h basis becausethere was not enough time to completethe assayson a 3-hourly basis.Before pooling for 3-h 24-h to eight urine samples give one sample eachof the 7- and 9- week-old gerbils, 200 pl from each3-h urine sample was pipetted into Eppendorf tubes and stored at -20*C. The pooled 24-h urine sampleswere diluted 1:10 with buffer. Procedure

The assaysize was limited to about 160 tubes to avoid assay drift. The volumesrequiredin the assaywere: Sample/standard Antiserum. Label Dextran-coatedcharcoal Total volume

500 PI 200 pI 100 PI 100 PI 900 PI

The aMT6s standards were made up in 1:25 CSU and 500 pl (See dispensed form the to page 78). standard curve. were Unknown samples, NSBs, and standards were pipetted into LP3 tubes (Luckhams, Burgess Hill, Sussex,UK) in duplicates using Gilson pipettes or repeating Eppendorf microlitre dispensers; zero tubes were prepared in quadruplicate. 200 pl of antiserum.at a final dilution of 1: 40 000 was added to all the assay tubes except the total and NSB, tubes. The total tubes only contained 100 pl label so that the total radioactivity of the label could be counted. The NSB tubes received 200 pI tricine

86

buffer instead of 200 gl antiserumto discover whether or not other compounds bind with the antigen. The tubes were vortexed for ten seconds and left to incubate at room temperaturefor 30 minutes. After adding 100 pl 1251-aMT6s(10 000 cpm), the tubes were for incubate left ten to vortexed seconds and overnight at 4'C. The bound- and free-aMT6s were separatedfrom each other by the addition of 100 pl DCC, (stirring at 4"C), to all tubes for After the totals. ten seconds, the tubes except vortexing left incubate for 15 minutes at 4"C and then centrifuged to were at 2500 rpm for 15 minutes at 4"C. Immediately after centrifugation, the supernatantswere discarded. The radioactivity in the charcoal pellets, (the free 1251-aNff6s antigen), was counted for 100 seconds in a 12 channel gamma UK). Surrey, (Multigamma, Selsdon, LKB Ltd, counter Standard curves were plotted on B/BO% (y)/Iog(x) coordinates. The aNff6s concentration was determined from the doseresponsecurve using a'RIAcalc' program.

The 'RIAcalc' program determines the percentage of total counts (bound or free) and then plots them as a function of the known concentrations of the standards. A curve is fitted through the standard points using a smoothing spline calculation method. This fits a continuous curve to each pair of standard points using the least squaresmethod and it calculates the best fitting curve with a minimum number of turning

87

points. The concentrationsof urinary aMT6s in the unknown sampleswere determinedfrom the standardcurve. All samplesfrom individual gerbils at any particular age were analysed in the same assay. All analyses were done in duplicate. When the results did not agree within 10%, the sample was repeatedand the outlier was discarded. Samples which had aMT6s concentrationsgreaterthan 26 ng aMT6s/ml were repeatedusing a higher dilution. The excretion rate of aMT6s was calculatedby multiplying the concentration(ng/ml) with the correspondingurine volume. 3.6 Measurement of Melatonin Content in Gerbil Pineal Using Radioirnmunoassay

3.6.1 Collection of the Gerbil Pineals The subjects were 71 gerbils aged 16 weeks from: the HF females, (n (n 12 LF 12 the group group = males), = 12 females, 12 males) and an additional low-F group (LFNM) (n females, had been 12 II which = used as a pilot study males). Groups LF and HF consisted of gerbils whose urinary levels of aMT6s had been monitored at 7,9,11

Y2 and 16

weeks; group LFNM consisted of gerbils whose urinary levels of aMT6s had only been determined at 16 weeks of age. The 16-week-old gerbils from the LFNM, HF and LF groups were sacrificed in July, October and November, 1992,

88

in hours 48 day had the the they completed respectively, after metabolesfor the collection of urine. Six gerbils, (3 males and 3 females), from each group were intervals 24-h 6-h by dislocation over a sacrificed cervical at light); (3-h 2200 (during 1000 1600 the after the period: at and darkness). darkness) (9-h 0400 the onset of onset of after and The superficial pineal glandswere removedwith a minimum of delay, placed in 5 ml Eppendorf tubes containing a drop of dH20 and frozen rapidly on solid C02. During the dark phaseof the LD cycle, the sacrifice of the gerbils and the collection of dim from illumination the a pinealswere performedwith aid of light. The pinealswere storedat -20'C. red 3.6.2 Analytical Procedure

Melatonin concentrationsin the gerbil pineals were estimated by means of a sensitive and direct RIA that had been for (Fraser 1983; other species et al, previously validated Webley et al, 1985).Pinealswere homogenizedin I ml tricine buffer on ice using a glass pestle and mortar, (Glass Unit, University of Surrey). The homogenateswere transferred to LP3 tubes and centrifugedat 3500 rpm for 15 minutes at 4'C. The supernatantswere aspiratedand frozen at -20'C until time of assay. Estimation of the required volume ofpineal extractfor RIA The required volume of pineal supernatant for the RIA was estimated to ensure that the MT concentration lay within the

89

5-250 for MT: typically the pg/0.5 ml. range of standardcurve This was important becauseof the limited amount of available i. 500 for The RIA NIT e., ýLl sample. required one ml of sample in each duplicate. Assuming that the gerbil pineal produces about 50 pg MT/day, 200 pl of pineal supernatantcontains in buffer). homogenized I (each 10 MT ml about pg pineal was Assuming that the gerbil pineals contain higher MT levels during the night than the day, the following volumes of pineal supernatantswere used:-

night-time daytime

pineal supernatant 200 pI 300 pl

tricine buffer 300 pI 200 pl

3.6.3 Reagents

Water: All water was freshly double-distilledin glass. Tricine buffer.- Seepage77 Dextran-coatedcharcoal (DCQ: Seepage78 3H from Amersham, (Prod No. TRK-798, Label.. -melatonin International, Buckinghamshire, UK) was obtained in 0.25 mCi 3H-melatonin intermediate An was solution of quantities. label 2 by diluting 20 to of stock ml with absolute [LI prepared by diluting freshly The prepared ethanol. working solution was the intermediate solution with tricine buffer to give about 4000 in cpm 100 pl. Antiserum:

Sheep anti-melatonin

(Prod No. antiserum,

G/S/704-8483, from Stockgrand, Guildford, Surrey, UK). The antisera

were

raised

in

sheep

against

N-acetyl-5-

methoxytryptophan conjugated through the side-chain to

90

bovine thyroglobulin. It was supplied freeze-dried and was dilution intermediate d-H20 (an two of reconstitutedwith ml 1:10) and aliquoted into 50 pl portions and stored at -20"C. One 50 pl aliquot was diluted with 20 ml tricine buffer to give the working solution (initial dilution of 1:4000). Melatonin standards: Standardmelatonin (Prod No. M-5250, from Sigma Ltd). A stock melatonin standard was prepared in 0.5 by dissolving 10 three ml every mg melatonin months absoluteethanol and making up to 10 ml with d-1120to give a freshly I The concentrationof mg/ml. working standardwas for from this stock solution eachassay. prepared 100 pl of I mg/mI standard MT to 100 ml in d-H20 to give I pg/ml Nff. 0.5 ml of I pg/ml MT solution to 50 ml in d-H20 to give 10 pg, pI MT. 125 pl of 10 pg/pl Nff solution to 2.5 ml in tricine buffer to 0.5 give pg/pl MT. Further dilution of 0.5 pg/pI MT with tricine buffer provided the standard curve - 0,2.5,5,12.5,25,50,100 MT/0.5 ml as follows: Melatonin 0.5 pg/pl gl 0 5 10 25 50 100 200 500

Tricine buffer

250 and pg

Melatonin pg/0.5 ml

gl 500 495 490 475 450 400 300 0

0 2.5 5 12.5 25 50 100 250

91 f

Scinfillantfluid: The toluene-basedscintillant was preparedby 0.15 (Fisons) (PPO) 2.5 2,5-diphenyloxazole and g adding g 1,4-bis-(4-methyl-5-phenyl-2-oxazolyl) benzene (dimethyl POPOP, from Fisons) to 0.5 L toluene (low-sulphur grade, from Fisons).

3.6.4 Methodology

Except for the zero binding tubes, which were set up in NSBs totals were set quadruplicate, all standards, samples, and

in duplicate following the volumesof reagents:up with Samples/standards Antiserurn Label Dextran-coatedcharcoal Total volume

500 PI 200 pI 100 PI 500 pI 1300pI

500 pI sample(200 pI pineal supernatantand 300 pl buffer, or 300 pl pineal supernatantand 200 pI buffer, dependingupon dayduring taken the the night-time or whether sample was time respectively), MT standard or QC were added to each duplicate assaytube. 200 pI antiserum.was addedto all tubes for The 10 NSBs. tubes the totals vortexed were except and for incubate 30 minutes at room left to seconds and 3H-melatonin After 100 temperature. vortexing, pl was added. the tubes were incubatedfor 18 hours (overnight) at 4'C. 500 from MT DCC 4'C to pl at was added separateantibody-bound free MT. The tubeswere vortexed,incubatedfor 15 minutesat 4"C, and centrifugedat 1500rpm for 15 minutesat 4*C. 700 pl

92

aliquots of the supernatants(containing the antibody-bound fraction) were pipetted into vials containing 4 ml scintillant fluid. The vials were thoroughly shakenfor one hour at room 3H-melatonin into the in temperature order to extract all the for is This organic phase. system a more efficient medium radioactivity detection than a detergent-basedsystem and is lesssensitiveto quenching. The B-radioactivity in the tubes was counted for 5 minutes using

an

automatic

B-scintillation

counter

(Rackbeta,

Pharmacia). The counting efficiency of the samples was 25%. The sample concentrations were calculated using a 'RIAcalc' program.

3.7 Physiological Signs of Sexual Maturity in Gerbils

The areas of the ventral glands: At 10,16 and 28 weeks of age, the widths and lengths of the ventral glandswere measuredto the nearest 0.5 mm using callipers. The areas of the glands were length for by formula (1/4pi the estimated x x width). To an ellipse facilitate measurementof the ventral gland,the abdominalhair was dampenedwith a small amountof water. The age at vaginal opening: Each female was examineddaily for vaginal opening. The age at which vaginal oestrousoccurredwas recorded. Body weights of gerbils were recorded(to the nearestgram). Right and left testes were removed and weighed at time of in data Combined the testes sacrifice. used were weights of analysis.

93

3.8 Statistical Methods

3.8.1 The Fluoride Contents of Human Tissues The [F] of pineal (wet), muscle (wet) and bone (ash) and [Ca] (wet) SD. The amount of + of pineal were expressedas mean hydroxyapatite (HA), CaO(PO4)6(OH)2, in the pineal was determined as follows:- MW of HA = 1004, therefore HA = total calcium x 1004/400,HA = total calcium x 2.5. The [F] of HA was expressedas mean± SD. The degreeof calcification was determinedby expressingthe weight of pineal HA as a percentageof the original weight of pineal. Statistical differences between the mean [F] in pineal and Student's (wet determined muscle using unpaired weight) were Mest (two tailed). PearsoWscorrelation coefficients were used to test correlationsbetween:i) pineal [F] and pineal [Ca]; bone degree iii) [F] [F]; of ash pineal weight and and pineal calcification. Levels of significancewere set at p

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Table C.25 Laboratory resultsof circadian excretion of urinary aMT6s over 24-h from low-F female gerbils aged 16 weekson 8.11.92. QCs: 3.5,14.3,19.5 TIME

16WLF F1

16WLF R

16WLFF3

16WLF F2

volume &MT6s aMT6s volume aMT6s aMT6s volume aMT6s aMT6s volume aMT6s &MT6s ng/ml us ml ng/ml Rg ml OgIml a& ag/ml ng ml rat

1800-2100

14.6

1.315

1.9

8.2

6.875

5.6

10.8

0.760

0.8

12.9

1.249

1.6

2100-2400

7.5

6.036

4.5

7.9

6.819

5.4

8.5

2.259

1.9

13.4

1.965

2.6

2400-0300

13.9

0.315

0.4

11.3

0.316

0.4

9.5

2.581

2.5

9.3

0.285

0.3

0300-0600

9.6

9.972^

19.1

9.6

12.891

12.4

12.7

3.084

3.9

13.0

0.314

0.4

0600-0900

6.9

0.314

0.2

9.8

10.088

8.9

15.3

1.157

1.8

16.9

12.708 21.5

0900-1200

8.9

5.663

5.0

8.2

3.912

3.2

6.1

0.355

0.2

6.8

0.295

0.2

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11.1

0.285

0.3

11.3

0.428

0.5

9.2

3.841

3.5

12.0

3.708

4.4

1500-1800

9.4

S.135

4.9

8.2

4.764

3.9

6.3

0.352

0.2

14.4

2.359

3.4

36.4

40.2

14.9

34.4

body weight g

68

61

63

77

aMT6s pgIg BW/24-h

537

657

236

447

total aMT6s ng/24-h

QCs: 3.5,14.3,19.5 TIME

16WLF F5 volume aMT6s UgImI ml

16WLF F6

&MT6s volume &MT6s &MT6s volume Offlis at

ml

16WLF FS

16WLF F7

ag/ml

at

ml

aNff6a

ng/ml

at

volume SNIT62 aMT6s ml

ag/ml

at

1900-2100

12.6

1.136

1.4

13.4

2.552

3.4

14.4

1.369

2.0

10.9

0.333

0.4

2100-2400

8.7

1.739

1.5

10.2

2.022

2.1

9.5

2.692

2.6

7.3

2.922

2.1

2400-0300

9.5

4.071

3.9

10.2

5.312

5.4

10.4

7.798

8.1

13.2

7.173

9.5

0300-0600

10.5

13.506

14.2

10.1

6.382

7.1

9.6

15.910 15.3

12.4

3.989

4.9

0600-0900

9.6

3.370

3.2

11.3

2.219

2.5

11.4

2.861

3.3

6.3

0,599

0.4

0900-1200

9.3

4.325

4.0

8.3

3.812

3.2

9.9

2.209

2.2

12.9

1.800

2.3

1200-1500

13.0

1.472

1.9

9.8

1.053

1.0

14.5

1.123

1.6

16.2

2.312

3.7

1500-1800

7.9

6.146

4.9

11.1

3.513

3.9

8.6

3.016

2.6

5.4

0.375

0.2

35.0

28.6

37.6

23.6

body weight g

75

69

68

75

aNff6s pgIg BW/24-b

469

412

554

315

total aNff6s ngI24-h

QCs: 3.8,14.9,25.4 TIME

16WLF F9

16WLF FI I

16WLF FIO

16WLF F12

volume aNff6s &MT6s volume aMT69 &Nff6s volume &Nff6s aMT6a volume aNff6s aMT6s MI DgIml ng MI as ml ag/ml at us ng/ml MI ngIml

1800-2100

19.1

0.664

1.2

9.8

3.470

3.4

13.5

1.566

2.1

13.0

3.350

4.4

2100-2400

6.7

1.734

1.2

7.9

1.849

1.5

9.7

0.259

0.3

5.8

1.521

0.9

2400-0300

17.1

4.556

7.8

11.4

3.359

3.9

11.4

4.318

4.9

19.4

0.727

1.4

0300-0600

6.5

13.000

8.5

3.6

0.294

0.1

12.3

12.811

15.8

10.5

2.992

3.0

0600-0900

2.7

0.221

0.1

8.3

14.798

12.3

2.5

0.583

0.1

12.9

1.380

1.8

0900-1200

12.9

3.764

4.9

12.6

4.088

5.2

20.0

2.604

5.2

14.6

0.741

1.1

1200-1500

11.6

2.354

2.7

12.6

0.316

0.4

6.8

0,320

0.2

6.3

3.733

2.4

1500-1800

14.7

1.701

2.5

17.4

2.241

3.9

11.9

2.698

3.2

9.5

0.782

0.7

28.8

30.5

31.8

15.6

body weight g

63

64

62

66

aMT6s pgtg BW/24-h

457

481

513

239

total aMT6s ng/24-h

229

Table C.26 Laboratory results of circadian excretion of urinary aMT6s over 24-h from high-F female gerbils aged 16 weeks on 1.10.92. QCs:3.6.13.2,23.1 TIME 1600- INK) 1900-2200

16WHF F1

16WHF F2

16WHF F3

16WHF R

volume&MT6saMT6svolumeaMT6saMT6svolume&MT6saMT6svolumeaMT6saMT6s ml ng/ml ng ml ng/ml ng ml ng/ml ng ml ng/ml ng 6.6 0.347 2.3 10.0 0.254 2.5 9.8 0.288 2.8 12.5 0.022 0.3 6.8

0.295 2.0

11.5 0.022 0.3

10.0 0.261 2.6

9.1

2200-0100

11.9 0.037 0.4

14.5 0.182 2.6

13.7 0.140 1.9

16.7 0.024 0.4

0100-0400

9.2

1.133 10.4 11.5 0.021 0.2

10.8 0.676 7.3

16.5 0.357 5.9

0400-0700

14.4 0.759 10.9 11.3 0.948 10.7 19.7 0.664 13.1 14.2 0.845 12.0

0700-1000

8.4

0.036 0.3

11.8 0.317 3.7

11.7 0.037 0.4

8.2

1000-1300

10.1 0.453 4.6

12.5 0.028 0.3

10.6 0.332 3.5

10.5 0.383 4.0

1300-1600

7.9

7.9

11.6 0.143 1.7

7.6

0.024 0.2

0.488 3.9

0.413 3.8

0.026 0.2 0.025 0.2

totalaMT6sng/24-h bodyweightg

31.1

24.3

33.3

26.8

69

66

69

70

aMT6spg/gBW/24-h

449

371

481

380

QCs:3.8,13.7,22.5 TIME 1600-1900

16WHF FS

16WHF F6

16WHF F7

16WHF FS

volume&MT6saNlUs volumeaMT6s&MT6svolumeaNlT6zaMT6svolumeaMT6sWTU ml Ug/ml ng ml ng/ml ng ml ng/ml us ml ng1ml ng 11.2 0.133 1.5 8.5 0.037 0.3 7.8 0.316 2.5 13.8 0.090 1.2

19W- 2200 2200-0100

10.5 0.070 0.7

12.2 0.052 0.6

10.5 0.009 0.1

11.5 0.128 1.5

16.4 0.017 0.3

8.2

0100-0400

12.8 0.072 0.9

16.6 0.071 1.2

12.1 0.103 1.2

11.9 7.448^ 17.7

0400-0700

16.1 0.372 6.0

18.9 0.088 1.7

9.5

11.8 0.014 0.2

0700-1000

10.9 0.118 1.3

9.8

10.2 7.908^ 16.1

9.2

1000-1300

4.5

0.430 1.9

13.2 0.056 0.7

8.3

0.727 6.0

17.5 0,013 0.2

1300-1600

9.4

0.130 1.2

8.8

5.2

0.017 0.1

6.0

0.140 1.4

0.140 1.2

12.2 0.138 1.7

1.215 10.0 14.5 0.011 0.2 0.010 0.1

8.46^ 15.6 0.573 3.4

totalaMT6sng/24-h bodyweightg

15.0

7.4

36.1

40.2

58

49

91

70

&MT6spg/gBW/24-h

258

153

398

572

QCs:3.6,12.6,22.1 TIME 1600-1900

16WHF F9

16WHF FlO

16WHF Fl.I

16WHF F12

volumeaNtUs aMT6svolumeaMT6saMT6svolumeaMT6saMT6svolumeaMT6s&MT6s ml ng/ml ng ml ngtml ng ml ng/ml ng ml ng/ml ng 11.8 0.030 0.4 12.1 0.101 1.2 11.8 0.079 0.9 9.8 0.004 0.0

1900-2200

10.8 0.046 0.5

21.0 0.041 0.9

14.2 0.060 0.9

20.5 0.033 0.7

22(W- 0100 0100- 04(W

12.1 0.029 0.4

10.0 0.579 5.8

14.5 0.162 2.4

12.4 0.007 0.1

13.7 0.034 0.5

10.5 1.259 13.2 19.1 0.333 6.4

17.3 0.200 3.5

0400-0700

18.3 0.080 1.5

9.2

0.986 9.1

10.1 0.478 4.8

7.8

0700-1000

11.5 0.157 1.8

7.3

0.399 2.9

5.3

17.5 0.392 6.9

1000-1300

8.4

0.025 0.2

8.1

0.004 0.0

17.7 0.149 2.6

9.6

0.225 2.2

1300-1600

8.4

0.239 2.0

9.2

0.331 3.0

6.7

4.4

0.004 0.0

0.221 1.2

0,014 0.1

0.004 0.0

totalaMT6sng/24-h bodyweightg

7.2

36.2

19.2

13.3

62

71

74

66

aMT6spg/gBW/24-h

115

511

262

202

230

Table C.27 Laboratoryresultsof circadianexcretionof urinary aMT6s over 24-h from low-F male gerbils aged 16weekson 8.11.92. QCs: 3.9,15.3,25.5 TIME

16WLF MI

16WLF M2

16WLF M4

16WLF M3

volume &MT6s aMT6s volume jkMT6s &MT6s volume aMT6s &MT6s volume aMT6s &MT6s ml Dg/ml ug ml ug/ml ng ml ng ml ngtml ng Dg/ml

1800-2100

9.4

0.311

0.3

12.5

2.025

2.5

13.2

1.096

1.4

12.5

0.351

0.4

2100-2400

12.7

2.296

2.9

13.8

3.126

4.3

9.3

4.430

4.1

12.2

9.739

11.9

2400-0300

17.8

4.780

8.5

10.1

0.408

0.4

13.6

11.574

15.7

11.5

0.348

0.4

0300-0600

8.7

11.669

10.2

12.6

11.076

14.0

8.0

7.831

6.3

11.8

14.970

17.7

0600-0900

6.8

4.354

3.0

8.4

3.165

2.7

8.5

4.202

3.6

10.5

12.452

13.1

0900-1200

10.0

0.449

0.4

9.4

2.877

2.7

5.3

0.392

0.2

12.1

0.816

1.0

1200-1500

7.0

5.788

4.1

136

1.716

2.3

9.8

5.283

5.2

12.7

2.770

3.5

1500-1800

12.4

2.950

3.7

12.1

1.987

2.4

15.8

1.613

2.5

9.6

5.616

5.4

33.0

31.3

39.1

53.4

body weight g

88

82

88

82

aMT6s pg/g BW/24-h

377

384

446

652

total *MT6s ng/24-h

QCs: 4.0,14.1,23.2 TIME

16WLF MS

16WLF M6

16WLF M7

16WLF NIS

volume aMT6s &MT6s volume aMT6s &MT6s volume aMT6s aMT6s volume aMT6s aMT6s ml ag/ml ag ml ug/ml ng ml Dg1ml ag ml ug/ml ug

1800-2100

19.3 0.285

0.6

15.0 0.878

1.3

15.3

1.913

2.9

13.7

1.706

2.3

2100-2400

11.4 4.113

4.7

10.5

1.021

1.1

11.2

4.285

4.8

11.3

3.594

4.1

2400-0300

9.2

8.125

7.5

11.2

1,980

2.2

11.7

9.256

10.8

13.0

9.944

12.9

0300-0600

9.7

14.075 13.7

5.9

4.586

2.7

7.9

22.447 17.7

10.6 22.010 23.3

0600-0900

7.6

5.065

3.8

11.5

1.474

1.7

10.4

5.607

5.8

8.1

4.443

3.6

0900-1200

11.0

1.425

1.6

12.4

1.001

1.2

12.9

0.530

0.7

12.7

2.110

2.7

1200-1500

9.9

2.602

2.6

13.6

1.467

2.0

11.2

4.889

5.5

10.5 2.548

2.7

1500-1800

11.0 2.964

3.3

11.2

1.065

1.2

8.3

7.074

5.9

16.6

4.8

2.884

37.6

13.4

54.2

56.4

body weight g

81

74

108

91

aMT6s pg1gBW/24-h

464

181

502

617

total &MT6sng/24-h

QCs: 3.7,14.1,26.5 TIME

16WLFM9

16WLFMIO

16WLFMII

16WLFM12

volume aMT6s &MT6s volume &MT6s aMT6s volume aMT6s aMT6s volume aMT6s aMT6s ml ng1ml ng ml ngtml ng ml ug/ml ng ml ng/ml ng

1800-2100

15.2

2.252

3.4

18.2

1.435

2.6

10.1

3.272

3.3

16.6

1.984

3.3

2100-2400

10.5 0.337

0.4

15.7

1.541

2.4

14.5

1.597

2.3

14.2 0.308

0.4

2400-0300

14.7 0.315

0.5

12.6

3.448

4.3

11.2

1.317

1.5

12.4

6.327

7.8

0300-0600

10.3

6.886

7.1

12.8

3.893

5.0

9.6

4.209

4.0

11.4

0.293

0.3

0600-0900

10.2

3.465

3.5

10.4 2.191

2.3

8.9

2.918

2.6

9.8

7.935

7.8

0900-1200

10.8

2.073

2.2

11.2 0.244

0.3

13.8

1.279

1.8

9.1

0.574

0.5

1200-1500

18.0 0.963

1.7

10.0 2.723

2.7

12.2

2.571

3.1

15.3

3.110

4.8

1500-1800

15.1

2.8

11.5

1.8

11.4

0.973

1.1

13.6

2.168

2.9

total aMT6s ng/24-h

1.830

1.606

21.6

21.5

19.7

27.9

body weight g

81

91

76

73

*MT6s pg1gBW/24-h

266

237

260

380

231

Table C.28 Laboratory resultsof circadianexcretion of urinary aMT6s over 24-h from high-F male gerbils aged 16 weekson 1.10.92. QCs: 3.8,14.9,25.4 TIME

16WHF M1

16WHF M4

16W]IF M3

16WHF M2

volume aNff6s aMT6s volume &MT6s aNIT6s volume aMT6s aMT6s volume aNff6s &MT6s Dg1ml ng ng ml ng ml lig/ml og1ml ng ml ng/ml ml

1600-1900

10.6

0.260

0.3

9.8

2.450

2.4

6.4

2.500

1.6

11.0

1.481

1.6

1900-2200

9.7

4.566

4.4

7.6

5.320

4.0

16.8

0.755

1.3

11.6

1.461

1.7

2200-0100

11.4

0.308

0.4

9.0

0.708

0.6

9.6

2.123

2.0

13.4

1.465

2.0

0100-0400

12.1

0.284

0.3

14.5

14.708 21.3

12.5

3.078

3.8

8.5

3.710

3.2

0400-0700

14.1

13.379

18.9

6.7

5.183

3.5

10.9

4.361

4.8

15.1

6.679

10.1

0700-1000

8.4

0.368

0.3

13.1

14.009

18.4

8.6

2.894

2.5

18.5

1.154

2.1

1000-1300

8.4

0.382

0.3

10.2

0.512

0.5

9.9

2.629

2.6

10.0

1.808

1.8

1300-1600

10.2

6.819

7.0

11.3

6.248

7.1

11.0

1.915

2.1

8.8

2.110

1.9

total &MT6s ng/24-h

31.8

57.8

20.7

24.3

78

86

75

69

407

671

278

351

body weight g &MT6s pg/g BW124-h QC's: 3.7,14.5,25.3 TIME

16WHF MS

16WHF M6

16WHF M7

16WHF MS

volume aNff6s aMT6s volume aNff6s aMT6s volume aNff6s aNff6s volume &MT6s aMT6s ml ug/ml ng Og ml ng/ml ng ml ug1ml ag ml og/ml

1600-1900

11.7

2.374

2.8

14.7

2.187

3.2

16.3

1.502

2.4

8.4

3.068

2.6

1900-2200

12.0

3.339

4.0

12.3

0.354

0.4

13.8

0.288

0.4

13.4

2.883

3.9

2200-0100

11.7

0.237

0.3

10.7

4.283

4.6

13.0

2.492

3.2

9.6

0.348

0.3

0100-0400

12.2

4.276

5.2

11.1

0.319

0.4

8.9

12.402

11.0

10.2

9.910

10.1

0400-0700

10.8

12.419

13.4

15.4

3.546

5.5

8.7

6.649

5.8

9.9

15.102

15.0

0700-1000

9.9

2.406

2.4

10.4

2.601

2.7

13.8

0.519

0.7

9.3

3.648

6.1

1000-1300

10.6

3.520

3.7

15.8

1.378

2.2

13.6

2.200

3.0

9.4

0.459

0.4

1300-1600

8.9

0.371

0.3

6.3

0.510

0.3

8.9

3.031

2.7

8.3

6.027

5.0

32.1

19.3

29.3

43.3

body weight g

78

68

76

86

&MT6s pg1g BW/24-h

413

284

388

506

total aMT6s ng/24-h

QCs: 3.9,14.9,21.1 TIME

16WHF M9

16WHF M11

16WHF MIO

16WHF M12

volume aNff6s &MT6s volume &MT6s aMT6s volume aMT6s *MT6s volume aMT6s &Nff6s ml ng/ml og ng ml ngtml ug ml ng/ml ml ng/ml ng

1600-1900

13.5

0.379

0.5

12.5

4.215

5.3

11.0

0.059

0.1

17.6

0.751

1.3

1900-2200

15.0

2.732

4.1

12.0

3.146

3.8

12.4

2.678

3.3

14.6

0.048

0.1

2200-0100

8.8

0.390

0.3

9.1

0.040

0.0

12.9

2.003

2.6

13.6

4.751

6.5

0100-0400

10.0

4.397

4.4

14.8

9.121

13.5

9.6

0.041

0.0

10.1

0.051

0.1

0400-0700

12.4

9.834

12.2

12.2

0.050

0.1

13.4

8.706

11.7

14.5

14.047 20.4

0700-1000

7.6

0.551

0.4

13.2

4.994

6.6

12.4

1.276

1.6

10.6

9.058

9.6

1000-1300

11.6

2.962

3.4

6.1

0.042

0.0

4.8

0.065

0.0

8.8

0.111

0.1

1300-1600

9.9

0.409

0.4

8.9

4.972

4.3

12.1

3.099

3.7

9.6

0.047

0.0

25.8

33.6

23.0

38.0

body weight g

80

87

76

80

aMT6s pg/g BW/24-h

323

388

302

475

total aMT6s ng/24-h

1

232

Table C. 29 Laboratory results of urinary levels of aMT6s in female gerbils aged 1P/2 weeks over 48 hours in 2x 24-hour intervals. Shaded cells indicate where day I levels of aMT6s were used in statistical analyses. II '/2week high-fluoride female gerbils DAN'

QCs:

3.3,

13.0,24.3 I P/2W HF H P/2W

aMT6s ng/ml

I

DA N2

volume

aMT6s

ml

ng/day

aMT6s I

volume

ng/ml

aMT6s

MI

ng/day

aMT6s 2

ng/48-h

11.303

12.6

28.5

10.521 14.1

29.7

58.2

mean

body

aMT6s

wt

9

ng/24-h

aMT6s pg/g BW/24-h

29.1 )3.5 18.9

76 63

384 375

67

281

IIF

F2

9.617

12.0

23.1

7.974

15.0

23.9

47.0

IIF

F3

9.551

9.9

18.9

6.491

8.4

10.9

29.8

11 1/2W FIF F4

5.332

15.0

16.0

5.619

14.7

16.5

32.5

16.3

71

228

11 1/2W HF F5

5.492

17.5

19.2

11.828

11.9

7.771 7.334 7.386 6.403 6.595 5.616 5.922 4.494

16.5 10.5 18.5 24.3 18.9 14.8 16.5 17.7

25.6 15.4 27.3 31.1 24.9 16.6 19.5 15.9 1

4.553 6.204 7.355 7.755 9.746 5.549 5.385 6.89

28.2 22.9 20.3 30.9 32.9 34.5 20.4 18.7 18.9

47.4 48.5 35.8 58.2 64.0 59.4 37.0 38.3 34.8

23.7 24.3 17.9 29.1 32.0 29.7 18.5 19.1 17.4

70 68 61 58 55 63 51 48 57

337 355 295 498 580 475 362 397 303

1 P/2W I

1/2W FIF F6

11 HF 11 1/2W HF 11 I/zW HF 11 HF 11 '/, W FIF 11 P/2W HF 1 P/zW HF 1 1/2W

1/2W

F7 F8 F9 FIO F 11 F12 F13

25.1 16.4 21.0 21.2 17.7 16.7* 17.4 13.7

11V2week low-fluoride female gerbils DAY QCs:

3.8,12.9, 25.3

I

DAY I

aMT6s ng/ml

volume ml

2

I aMT6s ng/day

aMT6s I

Volume

ng/ml

aMT6s ng/day

aMT6s 2

ng/48-h

mean a

body

aMT6s

wt

9 -

II 1/2WLF F1 1/2WLF F2

11 1/2WLF 11 I/iWLF II V2WLF 11 11 1/ýWLF '/ýWLF

11 '/, WLF 11 '/ýWLF 11 1/2WLF 11 1/2WLF 11 1/2WLF 11 1/2WLF 11 1/2WLF 11 1/2WLF 11 1/2WLF 11 1/2WLF 11 1/2WLF 11

F3 F4 F5 F6 F7 F8 F9 FIO F 11 F12 F 13 F 14 F 15 F 16 F 17 F 18

11.582 14.4 8.188 20.7 5.788 18.5 4.156 18.4 4.966 22.0 8.091 19.8 9.958 12.5 8.343 16.0 5.397 14.2 8.651 12.5 8.615 14.9 12.402 10.8 7.946 16.4 7.526 16.7 10.407 10.1* 6.879 20.0 17.706 11.5 4.276 15.1

33.4 33.9 21.4 15.3 21.9 32.0 24.9 26.7 15.3 21.6 25.7 26.8 26.1 25.1 231 40.7 12.9

11.716 6.255 5.845 4.749 6.069 11.884 10.514 10.485 8.234 5.043 8.693 12.469 5.781 7.858 7.088 4.953 10.657 3.066

16.2 21.7 15.5 19.4 18.7 13.0 15.0 13.4 17.2 21.7 14.4 9.6 21.8 15.5 16.4 21.6 17.3 18.6

38.0 27.1 18.1 18.4 22.7 30.9 31.5 28.1 28.3 21.9 25.0 23.9 25.2 24.4 23.2 21.4 36.9 11.4

71.3 61.0 39.5 33.7 44.5

62.9 56.4 54.8 43.7

43.5 50.7 50.7 51.3 49.5 46.3 48.9 77.6 24.3

',5,7 33,9 21.4 16.9

22.3 31.5 28.2 27.4 21.8 21.8 25.4 25.4 25.6 24.7 23.2 27.5 38.8 12.2

56 56 57 60

56 54 55 65 61 58 66

63 65 59 58 65 54 59

aMT6s pg/g W/24

-h

633 607 373 281 400

578 509 423 356 376 383 406 394 417 397 426 725 205

233

Table C. 30 Laboratory results of urinary excretion of aMT6s in pubertal inale gerbils over 48 hours in 2x 24-hour intervals. Shaded cells indicate where day I levels ofaMT6s were used in statistical analyses. PUBERTAL IQCs: 3.8, 12.9,25.3 9 week hi g h-F males 9W HF MI 9W HF M2 9W HF M3 9W HF M4 9W HF M5 9W HF M6 9W HF M7 9W HF M8 11'A.week high-F males II 1/2WHFMI 1/2WHFM2 11 1P/2WHFM3 1P/2WHF M4 111/2WHFM5 111/2WHFM6 111/2WHFM7 111/2WHFM8 II 1/2WHF M9 1

aNIT6s ng/ml 7,198 4,309 7 108 17.001 7278 6073 6 782 5.188 aMT6s ng/ml 3,83 5 24 6499 8615 4361 4283 7401 10.412 9,192

HIGH-FLUORIDE

DAY I

DA N'2

%olume aNlT6s ml ng/day I

mean aMI'6s volume aNIT6s aNI'1'6s MT6s bodv Wt /g a pg ml ng/day 2 ng/48-h 9 BW/24-h ng/24-h

95 230 146 93 174 202 180 230

137 11)8 2o S 2-53 24 5 24.4 219

volume aMT6s ml ng/day I 20.5 187 137 100 17 1 165 160 17.0 18.1

15,7 196 178 172 149 i41 23.7 35.4 333

PUBERTAL QCs: 3.8,12.9, 25.3 9 week l ow- F males 9W LF MI 9W 11 M2 9W LF M3 9W LF M4 9W LF M5 9W LF M6 19WIT M7

aMT6s ng/ml 6.449 3 391 3997 2,966 3 546 1511 3.87

MALES

aNIT6s ng/ml 5.126 7.913 3.936 5 367 7 319 8402 6,275 9.432 aMT6s ng/ml 2.764 6 187 4929 3 594 3 851 4751 6 344 7 258 1)185

175 15.7 23,2 230 21,6 14,0 19.5 12.8

179 24.8 18.3 24,7 31.6 23.5 24.5 24.1

31 6 447 390 563 569 48 ) 489 48.0

15.8 22 3 195 31.6 28 5 240 24.4 240

62 65 53 64 55 56 68 70

255 346 371 491 521 426 360 345

mean aMT6s body wt volume aMT6s aMT6s MT6 S : pg/g ml ng/day 2 ng/48-h 9 BW/24-h g/24-h 25.7 179 170 22,9 250 14A 12.6 130 145

LOW-FLUORIDE

14.2 22,1 168 165 193 13.7 16.0 18.9 26.6 1

29,9 41 7 346 33 7 342 27 9 397 543 59.9

150 209 17 3 168 17 1 11C) 23.7 35.4 33.3

62 68 62 59 62 68 67 69 71

242 306 279 284 275 204 352 515 1 467

MALES

DAY I

DAY 2

volume aMT6s mi ng/day I

mean body aMT6s volume aMT6s aMT6s wt MT6 /g : s pg ml ng/day 2 ng/48-h 9 BW/24-h g/24-h

120 21A 21 8 16.8 21 5 22 3 MO

155 145 174 Mo 152 112 155

aMT6s ng/ml 4447 4,062 4.621 3,841 4687 3,683 3 517

156 16 5 23 1 24 5 IT2 205 22 1

139 147 21 3 188 16 1 15 1 15 5

294 29 3 38 8 28 8 31 4 26 3 31 0

147 146 194 144 157 13 1 15,5

67 62 63 61 59 54 67

219 237 309 238 271 244 230

234

Table C. 31 Laboratory results of urinary levels of aMT6s excreted by 16-week-old gerbils over 48-hours in 2x 24-hour intervals. Shaded cells indicate where the day I levels of aMT6s were used in statistical analyses. 16 week low-F females DAY2

DAN' I aNIT6s ng/ml

volume ml

aMT6s ng/day I

aMT6s ng/ml

volume ml

16W LF FI

3.756

17.8

13.4

3.789

18.0

13.6

27.0

mean aMT6s ng/24-h 13.5

16W LF F2

2.072

30.3

12.6

3.089

22.6

14.0

26.5

16W LF F3

5.528

16.2

17.9

4.290

20.2

17.3

16NV LF F4

6.534

1ý. g

20.6

16W LF F5

4.501

16,7

15.0

-1.299 2.271

28.5

16W LF F6

4.661

17.2

16.0

2.723

QCs: 3.8,13.1, 24.7

aNIT6s aMT6s ng/day 2 ng/48-h

aNI'1*6s body wt pg/g 9 BW/24-h 71

199

13.1

60

221

35.2

170

67

265

13.1

33.8

20.6

68

305

15.0

6.8

21 8

15.0

SI

286

24.5

13.3

29A

16.0

61

263

16 week high-fluoride females DAY QCs: 3.8,13.1, 24.7

I

DAY2

aMT6s ng/ml

volume ml

aMT6s ng/day I

aMT6s ng/ml

volume ml

16W FIF F1

7.674

12.7

19.5

4.571

18.4

16.8

16W HF F2

4.862

24.8

24 1

5.555

19.4

21.6

-, 45.7

16W HF F3

3.368

20.8

3.043

19.5

11.9

16W FIF F4

6.317

22

9.498

17.2

32.7

1

2- 8

aMT6s aMT6s ng/day 2 ng/48-h 36.

mean aNIT6s ng/24-h 19.5

aMT6s body wt Pwg 9 BW/24-h 76

256

22 8

59

387

25.9

14.0

65

216

60.5

3 ().2

71

426

16 week low-fluoride males DAY QCs: 3.4,14.2, 24.2

I

DAY 2 mean aMT6s ng/24-h

aMT6s body wt PWg 9 BW/24-h

aMT6s nWml

volume ml

aMT6s ng/day I

aMT6s ng/ml

volume ml

16W LF MI

4.172

10.3

8.6

6.905

16.8

23.2

31.8

15.9

74

215

16W LF M2

7.771

13.7

2 1.3,

6.255

17.7

22.1

43.4

21 11

79

278

8.3

3.769

17.6

13.3

31.5

18.3

79

231

14.5

7.412

17.7

26.2

40.7

20-1

71

287

20.2

3.851

22.8

17.6

37.9

18.9

81

233

28.2

7.760

10.3

16.0

44.2

28.2

77

367

77

287

70

207

16W LF M3

7.198

12.7

16W LF M4

5.171

14.0

16W LF M5

5.619

18.0

-

F1 r_

aMT6s aMT6s ng/day 2 ng/48-h

16W LF M6

7.847

18.0

16W LF M7

3.792

lost

6.723

16.4

22.1

16W LF M8

4.644

15.6

2.531

21.7

11.0

25.5

16W LF M9

6.516

16.0

5.823

18.0

21.0

41.8

20.9

-1 1

67

1

312

16 week high-fluoride males DAY

I

DAY 2 I

aMT6s nWmI

volume mi

aMT6s ng/day I

aMT6s ng/ml

volume ml

16W HF MI

5.703

15.0

17.1

7.579

16.5

25.0

42.1

mean a NIT6s ng/24-h 21.1

16W FIF M2

7.946

20.6

5.807

20.8

24.2

56.9

16W HIF M3

6.200

19.1

23.7

5.769

25.0

28.8

16W HIF M4

7.228

18.5

26.7

9.288

19.2

16W HF M5

8.211

20.5

33,7

7.722

16W HF M6

7.489

20.0

30.0

16W FIF M7

5.807

21.0

116W HF M8

1 5.619

25.1

QCs: 3.4,14.2, 24.2

aNIT6s aMT6s ng/day 2 ng/48-h

aMT6s body wt PWg 9 BW/24-h 80

263

32.7

79

414

52.5

26.3

96

305

35,7

62.4

31.2

71

440

22.5

34.7

68.4

34.2

77

444

6,763

15.5

21.0

50.9

30.0

71

423

24.4

1912

23.7

18.5

42.9

24.4

65

375

28.2

4.697

22.6

21.2

1 49.4

28.2

70

403

235

Table C. 32 Laboratory results of urinary levels of aMT6s in female gerbils aged 28 weeks over 48hours in 2x 24-hour intervals. Shadedcells indicate where the day I levels of aM,F6s were used in statistical analyses. 28 week high-fluoride females DAN' I

QCs: 3.6,13.1,21.3

a,NIT6s nWml

DAY2

volume mI

aNlT6s ng/day I

aNIT6s ng/ml

volume mI

aNIT6s ng/day 2

aMT6s ng/48-h

mean

a NIT6s ng/24-h

aM T6s

body wt pWg 9 BW/24-h

28W HFF1

6275

13 5

169

6023

160

19 3

362

18 1

73

247

28W HF F2

6275

19,0

23.8

6.344

21.7

27 5

51.4

25 7

o4

401

28W HF F3

4.656

17.5

163

8.976

9,9

17 8

34.1

170

79

219

28W HF F4

6.033

14A

174

3 736

21.6

16 1

33 5

16 8

78

215

28W HF F5

5.292

2&9

22 1

6.849

146

200

42.1

21 1

74

286

28W HF F6

7.123

149

21,2

6.33

20.5

260

47.2

23.6

68

345

28W HF F7

4.389

15,2

13 3

3,794

230

175

30.8

154

72

213

28W HF F8

4,551

14.3

13 0

7 831

15.4

24.1

37.1

186

81

228

28W HF F9

5,489

16.6

18 2

5607

17,1

192

374

18 7

76

248

11.235 M751 4ý902 9275

F, 7, jj, 14.0 ý 156 335 155 Is 2 135F 25.0

8 543 8ý567 4328 6,782

15,0 18.9 22.7 14,0

256 32.4 196 190

571 _31.5, 330 65,9 348 174 25.0 440

78 73 78 89

405 450 223 280

28WHFFIO 28WHFF11 28WHFF12 28WHFF13

28 week low-fluoride females DAY

aMT6s ng/ml

volume ml

28W LF F1

6.036

28W LF F2

DAY 2

I

aMT6s ng/day 2

aMT6s ng/48-h

mean aMT6s ng/24-h

19.5

292

47,9

23 9

68

353

4 511

20.8

M8

36 5

19 3

67

272

Ii. 9

2,890

21 5

12,4

27 3

14.9

62

239

1-19

3 970

2L2

168

29 7

141)

64

231

14.1

2 382

17.8

&5

22

14.1

73

194

212

4.707

21.0

19.8

421)

23.2

73

319

aMT6s ng/day I

aMT6s ng/ml

15ý5

18 7

7479

3 83

23.2

17 8

28W LF F3

193

254

28W LF F4

3.978

16.2

QCs: 3.6,13.1,21.3

volume ml

aMT6s

body wt pg/g 9 BW/24-h

28W LF F5

3.398

207

r

28W LF F6

6.099

190

1

28W LF F7

4.87

15,2

14 8

4206

18 3

15 4

302

1ý 1

71

214

28W LF F8

4934

14 5

143

T360

14 3

21 0

35 4

17 7

72

244

28W LF F9

2 217

16.5

73

3.325

166

11 0

184

92

68

135

28W LF FIO

2 762

19,0

10 5

1663

18 2

13 3

23 8

11 9

70

171

28W LF F11

4.306

18A

15 8

4,591

20.5

18 8

34.7

17 3

69

253

28W LF F12

4.613

21 0

194

6.845

14.4

197

39 1

19 5

72

272

28W LF F13

7,083

15.5

3,274

19.0

12 4

34 4

22.0

79

277

4445

21A

23,4

25 2

22 1

75

294

28W IT F14

190

15

379

1

44 2

F

236

Table C. 33 Laboratory results of urinary levels of aMT6s in male gerbils aged 28 weeks over 48-hours in 2 x 24-hour urine collections. Shaded cells indicate where the day I levels of aMT6s were used in statistical analyses. 28 week high-fluoride male gerbils DAV

I

DAV2

aMT6s ng/day 2

aMT6s ng/48-h

mean a M '1'6s ng/24-h

13.9

32.7

72 3

39.6

93

427

126

30,2

546

27 1

95

286

25 4

11 ý981 12 128

15 2

369

62,3

31 1

97

358

17.0

28 1

8,834

15,0

265

54.6

273

83

329

3936

15 7

124

4.174

18 5

154

27.8

13 9

86

162

28W HF M6

5974

173

20,7

9.172

140

25.7

46.4

23 2

97

240

28W HF M7

9314

15.3

28,5

5 220

24.6

25,7

54.2

27.1

103

263

28W HF M8

6.570

20.6

2TI

8.927

21 4

A2

65 31

32.6

aMT6s ng/ml

volume ml

2MI I6s -F/day I

28W HF MI

10,150

195

39.6

11,764

28W HF M2

9903

12 3

24 4

28W HF M3

8,472

15,0

28W HF M4

8274

28W HF M5

QCs: 4.1,14.6,21.7

aMT6s ng/ml

1

volume mi

aMT6s body wt pg/g 9 BW[24-h

1

85

1

384

28 week low-fluoride male gerbils DAY

I

DAV 2 mean

aMT6 S ng/day 2

ng/48-h

aMT6s ng/24-h

15.5

IT9

406

22.7

113

202

4646

197

183

37 1

19 5

108

172

ITO

7 897

16.6

26.2

43 2

21 6

106

204

15,0

13,6

5.403

22.5

243

379

189

114

166

6 173

20.8

25 7

6.099

236

28.8

54ý5

27 2

73

373

28W LF M6

7.396

200

296

9373

18,2

34,1

63.7

31 9

84

379

28W LF M7

7760

17,4

T422

13 4

19.9

469

27.0

77

350

8,449

14.0

23 7

444

22 2

109

203

6248

22 8

2&5

59 5

29 8

72

413

aMT6s ng/ml

volume ml

ng/day I

aMT6s ng/ml

volume ml

28W LF MI

6.606

172

22.1

5 775

28W LF M2

4,689

20.0

18 8

28W LF M3

4.880

17.4

28W LF M4

4,526

28W LF M5

QCs: 4.1,14.6,21.7

aMT6s

28W LF M8

7,739

13 4

07 _T7ý 20ý7

28W LF M9

7 218

21 5

31 0

aMT6s

aMT6s body wt Wit 9 BWt24-h

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-ý llý Cý rý . 12' r, '-It " en r- "I -7

14ý

wl

cq

C14

00 Cý

S'!

V. )

ýo

cli

CO A

rq

CA

-ý

C'i

_-;

--:

r-ý oý 0ý cq c, r-: c) cn ý

r-

rn

cn

r- :

.

"ý (0ý V, C) tr) r") "

-

-

(=

(=

-

cý

cn

en

--:

r-

m

m

0,

(2,

c"

r, 4 'n

'o

r-

C-)

--ý rý --ý --ý --: rý

Wý cn en c) rn C.)

6

6

-.

6 Cý -C-i ý-, ;,":

=

C.)

>

'10 u-

r--

tr)

C-4 W)

-,t

C'4

"

9

-

-0 -0 (7,

-

u

Cc

00

-

"

:2 "a

ca p w

2

2:

CD

rn

C-4 7:,

c 0 ýc 0,

--t

r-

c 7

fn

c 7

C,

C-4 Z;

-'T

C) -C) 0 C) c) C r.0) "o C) 0, C) cq C) 0 rq -'T r-

r-

cý

'n

ý

\.c

9

;,

-6b -ýb r_ r_ CD C) (= I. r(::, "o

cw

73

01) ýo

ý-

29

j-

v

.

.0 II

m

cl

co U

ý

00 00 m

-

0

'-ýz g ý20

00

cn

.4 4

W-ý

-,T

-

r-

"

(3, .,t -:

CD -

=2

fn (i

U

v

-0 =

_ I-73

_0

0 0 e .P -2

v

E

z

241

x w Ln

I",

Q

V'b ýo

e4

w 2

%0 rr4 M

oo cý r, 4 le

! -Z --

00

(Y,

Ir

en

rq

00

aý

10

-

r-

vi r-

n- 00

r-

mr

m)

cý

CY, 0, 00 4D 4 " V lb fn r.

r-

00

fn

No

0mG CD

CD

0 5

LM

cy, cy, wý r-4

-e o' mm

r, ;; r-%

--r, 2=

"0

cý

"

V'b 00 Ild" le

V'b

r-

r4

%0 "

vý

%Q

öo

Cb ,A

V. ý2 M-

2ýZ

f', '

%0 "

vl u

x u

eý ýk Z

ce c:

ý> CD c> 0 ýo r- v, c> Co m rn V.,oo W, C,4 m ON ýo ýo ýQ C,

M- -

0

0

-

9

r4 oo (> C-4

t r-

('4

tr)

"t

00

r-

t--

-t

C7,

"t

00

r-

c)

(=)

rc)

C)

C>

(D

,o

M C)

ý: ;

C)

-

,,

r14 kr)

00 CIA

rkf)

't CA

CN tn o ll C14

L4

llý I

C) rq

ýc V)

00 00

00

v)

r-

rn

-0 =

r-

03

.

m C> r-

r)

1" )

('4

(u

ý')

,*

cl)

kf)

C'A

"

M

:!

C,4

(, I

M

00

C: )

rl

f)

m

-

C) 00

kr

('4 rj 00 CN

C

cq

tf)

a,,

ID C) p t-ý , , tý, kn .

(11 J) >

00

u

kr)

C7,

Cý

't

00

"

Q)

..

0

E

V

rq

Lr)

-

k,()

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'T

ýo

kr) "o

C) "o

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cl )

cl)

It

"

t

00

c)

7

kti

)

ttIP-

C-)

r-

C'4

o" It

00 Cl )

=

4.

-0

0U to m

tlo

-ý4 U ý:

ý, I

U

4) ý: "o

LL.

If)

r-

-

Cq

4. -

cz

co

M

"

r-

r-

cf)

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14D "t

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IT

r-

Cl)

0

c)

C'4

Cýý

tr)

r-

00

00

00

C)

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kn

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m

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110

't

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00 "

fl)

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kf)

kf)

7, -

00 kl'l

"o

r)

00

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CD

00

6,

a,

ý,o

C)

CA

m

r -4

,ýý -

V-) ý(j ýu cl, (= 4 00 E V)

03

-

r- -0 C-4 == .2 ý C,

"a

(f)

r-

(71

kn

kn

r-

Cl)

L4

kf)

N

")

2 2

tf)

kf)

't

'IT

r-

IT

Cq

-t

r)

(: )"

kf)

C,4

C,4

Cl)

2

(,2

')

ý74

rr-

r-* 00 -,t C')

fl)

Cý c)

"t

kf)

C) IC

CD C) 0 Cl C) C)

C:) 0

CD 0 C) C)

C) (= CD C) "C

CD 0 CD C) (7, N

0 C:) c) C:) CD CD CD C> rC) "t -

1=1CI 0 0

C)

C)

0

c)

C) C) o C)

cz:)

C: 0 )

(=) 0

E I-

C::> kn C7, tr)

"

-a

ca

0 0 0 0 0 ON C'4

>, cz

1,0 In

:3

V%

-

0 t-

(=) CD C,) C)

0 C>

00 Ca t -0 ý: ý-, -0 ý -, ý' ý: ý M

0.

-

m m 0.

V)

cn 1.0

tA ý.c

M

M

m

0

0

0

C13 "o

*

rm u t-

CL

tn ý,o

-C

M tb

ý,c "

E

'o ,,

q) u

'0 a

a co

CD C)

4)

tt

U z V u X-

en

U Lu

A Q) M,

ON

C, 4 C-4"o m 00 7 CN CN

cz

1 ýZ 14Z

M Q

V

>,

ct

V -0

-0

ýu

00

ýE

03

u

a)

243

rl

00 Lo

r, 4

ýc

r, 4

C

cli

C, 4

rA

"o

r, 4

V)

z

rl

X

ýE

r-

CI A

ý'

-

*0

"')

c"

00

r,

rn

ýo rn

00 rn

qT rn

00 c)

rn

00

CD

m

m

(-, 1

W)

kt%

C,

kr)

M

rq

cc

'rT C-) C-4 -I-

03 c

Irl 'T

0

ý

q

rn

.0

ý7,

IT r-

'10

rA

7

rq

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q

ýC

-,t

00

T

::

kf)

'T

ý= rn

C, (N

ýc cl)

cr, -

C, rn

(7, (I

rr, 4

"T ,T

"C

(14 rl

00 rn

Lr)

rn

.C m

(:: )

00

.0

t--

00

r-

.0

0

rl V)

m

m

00

'n

-

kf)

r-

00

r, 4

") :r

,,

-.71-

cn

00

a.,

00

"C

V)

m

' 4-

V)

"o

tr)

M

C)

"T

"

00

M

V)

'It

"o

fn

rn

rn

V

"T r-

M C7,

00

r00

V) =

; ,

W)

"

00

a,

M c"I rl ) :T r) C'4 z: m rq :2 00

m -

r-

C , ,)

vu

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ej

rq

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V)

75 kl-ý

r-

0

Q)

ý:

-00 00 -,T (7, r- r- r)T r-

rl

r-

2

00 "

r-

C)

C)

00 rq

C)

IC

rW)

110

\0

C) C) C) m rý (= r-ý: c' ýý 0 2 - - :2 ý- 00 8, m 4

-ýn ýE $-

vto

ý:

rW-)

n

rl

-

C ', tri

>ý 00 ;00 00 Mra, " ) (11 "a

'ýz

"' ý ý

-0

7z =c

rq

ýý -

r ,4

-

c)

-

Irl

n

-

= ' :> " r4 N

ýR

N

"

r)

-

4

rA

1 4- N

"a ýo "0 "D ,T m ') .E !:2 cd

Cý

M

"

2ý :t

r-

"

n

C> C) W-) r00 r0 I.. W) tr) W)

C)

F=

>ý

ý. 0 m , - -0

ýu . ý:

"

421

; 16

vs

0

"t

A

> (N r ,4 -m

, C) 0 C) c) C) (=) 0 CD c) C, C) 0 0 c) C) C) 5 c) o 0 0 0 C14

ý;

't C)

r-

C)

(5 rn -

0ý cq " C) -.0 -

t-C:I.) C)

C) C) 5 "" m m m to

. . . 0. 0. . . . . . C:,. o. c). 0. C,. o. C) C> C) 0 CD 0 c) "1 7, :

-'T

:5

5 rq ý-

C)

5 rC)

(5 ::,

C)h

Eý) -6b

C! C! 110

V) ýo

v) ýo

Ll

0 CU C)

E

.0 ('3 Q

244

4)

rn 00

oc

't

rl)

ýc

X

r-)

00 r-j

r1i

OC

fl)

00

r-

It

00

Cý

2

r-

r-

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00

C)

C)

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"t

oc

4

IC

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00

ýc

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ON

CA

CIA

'IT

Cl)

(:::)

11,

Itt

W)

W)

C: ý

r--

r-

r) kf)

r14

rA

(14

C,

t.-)

r-

P-

r --, C)

(N 00

oc -

C) C)

rl

Cl)

rn

rA

oc 1,1 "o C> t00 C7, "o ON = "o , 00I C) V-1 qtl00 Ittýo cp, Itt "00 Q* ON C, "" C7, IV te) =5 ý: L, CIA rc) cl) 0,

0 C: o ' -

r-

-=7

r-

'r,

CIA "

V

-

CA

"

\C

110 1.0

E

::

In

t,

eq

'..

f-4 f-4 z

(4

"

=

ýd .z V-4 '"

C: )

ýo oll c)

:3 as

u U -0 a

kr)

kf)

V-)

")

rl rl (-1 "a2rI ;ý, 9m

(, A

C) C14

tf)

(14 It

ON 00

rIt

't clq

cl)

C7ý

r, 4

r)

-0 kr) W-)

rl) C, I

M

0

00

00

r')

r-)

r-

rq

eq

Nr

cN 00

tIT

r-

c)

11C Ol ýo

-At eq

17, ýo

17, ý10 4)

ý:

-

; 36.

\4

a,,

ýE =ý

cz

N

C) -)

M -

M -

r00

00

C) 00

r(14

'IT (14

IT It

rq It

01ý 110

C*l Nt,

Cý ý:t

r-

-

cu

\,c -

ýo "

00 -

00 -

11' C-4

fl) C-4

kn \C

IT M

t "

"o M

kn "o

rL7c r\0 -

(=) ttr) c)" cl)

C') \0

kn -

-

00 m

r-

m

C'4 'It

00 "t

a, c) m 0c) 00

ZT c)

cz

E

ý:ý=,; ý, "?

LL.

-

>ý

'ýz =ý

00

00

FN

C)

00

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C,4

kr)

kf)

-5=7 -

ca

-

(40

Cý

-

-

m

(-) "

(-I -

CIO

ýo

m

r"

oc -

r-

00 -

r-

V) -

It

m

kn

110 110 %%c -

Cl)

W)

"

W)

C14 "

"

00

r- tt

'It C',

F--

c) W-)

kn

0" C'\ C', 00 00 -

00

(71

(7,

00

t

00

Cf)

r1l

00

00

00

-

$u

-

" r, 4

ý: rco C)

A > Cd,

-> u

2-

4)

cl ,

00

ýc

It

', c C14

Cý r)

(") -T

"t c)

cl) :r

kf) t

1.0

(14

Itt

rr-

00 r-

!T 'T

'tt "tt

00

"

*

Cf)

"C

M

M

>, m

kil)

00 o"

C: C:

(14

=$ a

-

7) co

00

LIJ

; 7-

CD 0 O ý0 1

0 N

01 4 C

C>

C) CD

0

-

ql l

r-

Cý

0

0

I

I

C) 0 0

-

I

0 0

C) 0

fl)

ý10

-

-

-

I

0 C: ) ON

"

0 C>

I

I

0 0 It

-

(14 Cý

-

I

Cý 0

I

Cý

I

0 0

C: ) 0

0 0

1-

0

rn

C)

M

M

CL

m to -6b Cl CL

m

I

0 CD C) (::) 0 (= CD 0 CD c) g 0 C:) CD C:) 0 ý= C) 0 C) 0 "C CS rn rq C) C) fn I'D Cý CIA C> - 0"t r"t

0 o r- 0

1E

-9

-a cq

C

kr)

1

(U

'IT

-0 Q.) -0

I", "o "o 14)

ci a)

X

m

C. 'A

m

C13 m

C-4

(A

r-

g

L'a cz

r-

.'a

u

-

CA -0

=

245

Table C.42 Circadianexcretion of urinary aMT6s by low-F femalegerbils aged 16 weeks; correctedfor non-urinations;data expressedas ng/3-h. I

WINTERTIME

16W LF F1

16W LF F2

16W LF F3

16W LF F4

16W LF FS

16W LF F6

16W LF F7

16W LF F8

16W LF F9

16W LF FtO

16W LF F11

16W LF F12

1800-2100

1.9

5.6

0.8

1.6

1.4

3.4

2.0

1.2

1.2

3.4

2.4

2.2

2.3

1.3

0.4

2100-2400

4.9

5.8

1.9

3.3

1.5

2.1

2.6

1.3

1.2

1.5

2.5

0.9

2.5

1.5

0.4

2400-0300

9.6

6.2

2.5

7.2

3.9

5.4

8.1

9.5

7.8

3.9

2.4

1.4

5.7

2.8

0.8

0300-0600

9.7

6.2

3.9

7.2

14.2 7.1

15.3 5.3

8.6

6.2

15.9 3.0

8.6

4.4

1.3

0600-0900

2.6

8.9

2.0

7.3

3.2

2.5

3.3

1.2

2.5

6.1

2.7

1.8

3.7

2.4

0.7

0900-1200

2.7

3.7

1.8

2.2

4.0

3.2

2.2

1.1

2.4

5.6

2.7

1.1

2.7

1.3

0.4

1200-1500

2.4

1.9

1.9

2.2

1.9

1.0

1.6

3.9

2.7

1.9

1.6

2.4

2.1

0.7

0.2

1500-1800

2.4

2.0

1.3

3.4

4.9

3.9

2.6

1.2

2.5

2.0

1.6

1.4

2.4

1.1

0.3

8.1

2.3

5.5

1.6

totalaMT6sng/24-h

MEAN STDEV

36.2 40.3 16.1 34.4 35.0 28.6 37.7 24.7 28.9 30.6 31.8 14.2 29.9

Wy weight g

68

61

63

aMT6spg1gBW/24-h

532 661 256

77

75

447 467

63

69

68

75

64

414

554

329 459 478

62

66

68

SEM

513 215 444 126 36 1 1 1

Table C.43 Circadianexcretionof urinary aMT6s by low-F femalegerbils aged 16 weeks; correctedfor non-urinations;data expressedas pg/g BW/3-h. I

I

I

WINTER TIME

16W LF Ft

16W LF F2

16W LF F3

16W LF R

16W LF FS

16W LF F6

16W LF F7

16W LF F&

16W LF F9

16W LF FIO

16W LF Fit

16W LF F12

1800-2100

28

92

13

21

19

50

29

16

19

53

39

33

34

22

6

2100-2400

72

95

30

43

20

30

38

17

18

23

40

13

37

24

7

2400-0300

141 102

39

94

52

79

119 126

124

61

39

21

83

40

12

0300-0600

143 102

62

94

189 103 225

71

124

97

256

46

126

66

19

0600-0900

38

146

32

95

43

36

48

16

40

95

44

27

55

37

11

0900-1200

40

61

29

29

54

46

32

15

38

88

44

16

41

20

6

1200-1500

35

31

30

29

26

15

24

52

43

30

26

36

31

10

3

1500-1800

35

33

21

44

65

57

38

16

40

31

26

21 1 36 1 5 Hý6 4

415

552 329 446 477

513

237

i totalaMT6spg/gBW/24-h 533 661

256 447 467

MEAN STDEV

444

123

SEM

246

Table C.44 Circadian excretion of urinary aMT6s by high-F female gerbils at 16 weeks; corrected for non-urinations; data expressedas aMT6s ng/3-h. I

TIME

16W HF FI

16W HIF F2

16W HF F3

16W HF F4

16W HF F5

16W HF F6

16W HF F7

16W HIF F8

16W HF F9

16W HF FIO

16W UF I'll

1600-1900

2.3

2.8

2.8

2.1

1.1

0.3

2.6

1.2

0.4

1.2

0.9

0.4

1.5

1.0

0.3

1900-2200

2.4

1.4

2.6

2.1

2.2

0.6

5.0

1.9

0.5

0.9

0.9

0.4

1.7

1.3

0.4

2200-0100

5.2

1.4

1.9

3.0

2.2

0.3

5.0

9.0

0.4

5.8

2.4

1.8

3.2

2.6

0.7

0100-0400

5.2

5.3

7.3

2.9

3.0

1.2

1.3

8.9

0.5

13.2

6.4

1.7

4.7

3.8

1.1

0400-0700

11.2

5.4

13.5

12.2

3.0

1.7

8.0

7.9

1.5

9.1

4.8

3.5

6.8

4.1

1.2

0700-1000

2.3

4.0

1.7

2.0

1.3

1.4

8.1

7.9

2.0

2.9

1.2

3.4

3.2

2.4

0.7

1000-1300

2.5

2.0

1.8

2.2

1.9

0.7

6.1

1.7

1.0

1.5

2.7

2.2

2.2

1.4

0.4

1300-1600

2.0

1.9

1.7

1.4

1.2

1.2

2.5

1.7

1.0

1.5

1.5

0.9

1.5

0.4

0.1

16W HF MEA?s SD F12

SEM

38.6

40.2

7.1 36.1 20.7

14.3

24.9

11.8

3.4

48.6

90.8

70.3

62.1

70.7

73.5

65.9

68

9.9

2.9

153

425

572

115

510

282

216

356

147

43

total aMT6s ng/24-h

33.1 24.2 33.3

27.9

15.9

7.4

body weight g

69.4

65.6

69.4

70.4

58.3

aMT6s pg/g BW124-h 477

369

480

396

273

Table C.45 Circadian excretion of urinary aMT6s by high-F female gerbils at 16 weeks; corrected for non-urinations; data expressedas pg/g BW/3-h. I

I

16W HF F9

16W HF FIO

16W HF F11

18

6

17

13

6

55

27

8

12

12

6

55

129

6

82

52

24

14

127

7

174

52

34

88

113

25

29

22

28

89

30

26

31

33

15

29

29

24

20

21

480

367

483

399

259

TIME

16W HF F1

16W BF F2

16W HF F3

16W HF R

16W HF F5

16W HF F6

16W HF F7

16W HF F8

1600-1900

33

42

41

30

19

6

29

1900-2200

35

21

38

30

38

13

2200-0100

75

21

28

43

38

0100-0400

75

80

106

41

0400-0700

162

82

196

0700-1000

33

61

1000-1300

36

1300-1600 aMT6s pgtg BW/24-h

16W HF MEAls F12

SD

SEM

22

13

4

6

25

15

4

32

27

45

35

10

186

86

26

69

53

15

24

128

65

53

98

57

16

113

32

41

16

52

45

29

8

67

24

16

21

36

33

31

13

4

25

27

24

16

21

20

14

23

5

1

152

424

575

115

508

280 216 355

149

43

247

Table C.46 Circadianexcretionof urinary aMT6s by low-F male gerbils at 16 weeks;corrected for non-urinations;data expressedas aMT6s ng/3-h. I

WINTER TIME

16W 16W 16W 16W 16W 16W 16W 16W 16W 16W 16W 16W LF LF LF LF LF LF LF LF MEA11, SD LF LF LF LF M9 MIO Mll MI M2 M4 M5 M6 M7 M8 M12 M3

SEM

1800-2100

1.6

2.5

1.4

5.9

2.3

1.3

2.9

2.3

3.4

2.6

3.3

3.7

2.8

1.3

0.4

2100-2400

1.6

4.7

4.1

6.4

2.4

1.1

48

4.1

0.9

2.4

2.3

4.0

3.2

1.7

0.5

2400-0300

8.5

7.0

15.7 8.8

7.5

2.2

10.8 12.9 3.6

4.3

1.5

4.1

7.2

4.4

1.3

0300-0600

10.2 7.0

6.3

8.9

13.7 2.7

17.7 23.3 3.5

5.0

4.0

4.2

8.9

6.4

1.8

0600-0900

3.4

2.7

3.8

14.1 3.8

1.7

6.5

3.6

3.5

2.6

2.6

4.1

4.4

3.3

0.9

0900-1200

2.1

2.7

2.6

1.8

1.6

1.2

2.8

2.7

2.2

1.4

1.8

2.4

2.1

0.5

0.2

1200-1500

2.0

2.3

2.6

1.7

2.6

2.0

2.7

2.7

1.7

1.3

3.1

2.4

2.3

0.5

0.2

1500-1800

3.7

2.4

2.5

5.4

3.3

1.2

5.9

4.8

2.8

1.8

1.1

2.9

3.2

1.6

0.4

totalaMT6sngt24-h

33.0 31.3 39.1 53.4 37.6 13.4 54.2 56.4 21.6 21.5 19.7 27.9 34.1 14.5 4.2

bodyweightg

88

82

98

aMT6spg1gBW124-h

375 382 444 651 464

82

81

74

108

91

181 502 620

81

91

267 236

76

73

85

259

382 397

9.6

2.8

148

43

Table C.47 Circadianexcretionof urinary aMT6s by low-F male gerbils at 16 weeks;corrected for non-urinations;data expressedas pg/g BW/3-h. I

WINTERTIME

I

I 16WI 16WI 16WI 16WI 16WI 16W 16W 16W 16W 16W 16W 16W LF LF MEAls SD SEM LF LF LF LF LF LF LF LF LF LF MI

M2

M3

M4

M5

M6

M7

M8

M9

MIO

MI I

M12

1800-2100

Is

30

16

72

28

19

27

25

42

29

43

51

33

16.3

4.7

2100-2400

Is

57

47

78

30

15

44

45

11

26

30

55 , 38

19.9

5.7

2400-0300

97

85

179

107

93

30

100

142

44

47

20

56

83

46.9

13.5

0300-0600

116

85

72

109

169

36

164

256

43

55

53

58

101

66.0

19.0

0600-0900

39

33

43

172

47

23

60

40

43

29

34

56

52

39.4

11.4

0900-1200

i4

33

30

22

20

16

26

30

27

15

24

33

25

5.9

1.7

1200-1500

23

28

30

21

32

27

25

30

21

14

41

33

27

6.9

2.0

42

29

28

66

41

16

55

53

35

20

14

40

37

16.0

4.6

1500-1800 raUý6s 7pg/g BW124-h

375

382

444

651

464

181

502

620

267

236

259

382

397

1

148

1

42.8

248

Table C.48 Circadian excretion of urinary aMT6s by high-F male gerbils at 16 weeks; correctedfor non-urinations; data expressedas aMT6s ng/3-h. I

TIME

I

16W 16W 16W 16W 16W 16W 16W 16W 16W 16W 16W 16W HF HF HF HF HF HF HF HF HF HF HF HF MEAN SD M2 M3 M4 M5 M6 M7 M8 M9 MIO Mll M12 MI

1600-1900

2.2

2.4

1.6

1.6 2.8

1.6 2.8

1900-2200

2.2

4.6

1.3

1.7 4.3

2200-0100

6.5 12.3 2.0

2.0

0100-0400

6.5 12.3 3.8

3.2

0400-0700

6.5

9.2

4.8 10.1 13.4 2.7

0700-1000

2.3

9.7

2.5

2.1

2.4

1000-1300

2.3

3.6

2.6

1300-1600

2.3

3.5

78

86

SEM

2.6

2.0

2.7

1.6

1.4

2.1

0.5

0.2

2.3

1.6 4.2

2.1

3.8

1.7 3.2

2.7

1.2

0.3

2.6

2.7

1.6 5.0

2.2

6.8

2.6

4.1

3.1

0.9

2.6

2.8 11.0 5.1

2.2

6.8

5.9 10.2 6.0

3.5

1.0

6.5 15.0 12.6 3.3

5.8 10.2 8.3

4.0

1.2

2.7

1.5 6.4

1.7 3.3

1.6 9.7

3.8

3.0

0.9

1.8 3.7

2.2

1.5 2.5

1.7 2.2

1.9 2.0

2.3

0.7

0.2

2.1

1.9 2.3

1.5 2.7

2.5

1.5 2.1

1.8 2.0

2.2

75

69

68

86

80

76

78

3.3

0.6 1 0.2 totalaMT6sng/24-h 30.8 57.6 20.7 24.3 34.1 18.5 29.2 43.3 26.0 31.0 22.9 42.0 31.7 11.2 3.2 78

76

87

6.1

1.8

aMT6spgIgBW/24-h1 395 670 276 353 437 272 384 503 325 356 301 5251 400 1 118

34

bodyweightg

80

Table C.49 Circadianexcretionof urinaryaMT6sby high-Fmalegerbilsat 16weeks; for data expressed non-urinations; aspg/gBW/3-h. corrected TIME

I I I I I I I I I 16W 16W 16W 16W 16W 16W 16W 16W 16W 16W 16W 16W HF RF HF HF HF HF HF HF RIF HF HF HF MEA.Is SD SEM MI

M2

M3

M4

NIS

M6

M7

M8

M9

1600-1900

28

28

21

24

36

24

37

30

25

31

21

18

27

6

2

1900-2200

28

53

17

25

55

34

21

49

26

44

22

40

35

13

4

2200-0100

83

143 27

28

33

40

21

58

28

78

34

41

51

35

10

0100-0400

83

143 51

46

33

41

145 59

28

78

78

128

76

42

12

0400-0700

83

107 63

146 172 40

86

174 158 38

76

128 106

49

14

0700-1000

29

113 33

31

31

40

20

74

21

38

21

121

48

35

10

1000-1300

29

42

35

26

47

32

20

29

21

25

25

25

30

8

2

1300-1600

1 29

41

29

27

29

22

36

29

19

24

24

25

28

6

2

MIO M11 M12

aMT6spgIgBW/24-h 395 670 276 353 437 272 384 503 325 356 301 525 1 400 ý 118

34

249

Table D. 1 Descriptive statistics for urinary aMT6s levels by gerbils at 7 weeks.

Femilles

Mean StandardError Median Mode StandardDeviation Variance Kurtosis Skewness Range Minimum Maximum Sum Count Milles;

body weight

aMT6s pg/g BW/24-h

aMT6s ng/24-h

9

7W LF FEMALES

7W HF FEMALES

7W LF FEMALES

7W HF FEMALES

7W LF FEMALES

7W HF FEMALES

26.8 2.0 26.3 37.0 6.8 46.7

18.1 1.6 15.3 #N/A 5.5 30.1

602 49 545 NN/A 168 28361

360 32 292 #N/A 109 11901

-1.1 0.5 19.3 17.7 37.0 321 12

-0.4 1.1 15.4 12.2 27.6 217 12

-1.1 0.6 490 386 876 7221 12

-1.5 0.6 280 259 539 4314 12

45 0.9 45 42 3.0 9.2 4.6 1.9 11 42 53 538 12

51 1.6 53 53 5.6 31 0.3 -1.1 18 40 58 609 12

7W LF MAM

7W HF MALES

7W LF MALES

7W HF MALES

7W LF MALES

7W HF MALES

16.4 1.2 15.3 #N/A 4.2 18 7.0 2.4 16.5 12.0 28.5 197 12

566 42 576 #N/A 147 21533

308 22 306 #N/A 76 5753 3.2 1.1 311 185 496 3699 12

54 0.9 54 so 3.0 9

54 1.7 55 56.6 5.8 34

-1.1 0.1 9 50 59 649 12

-0.4 0.1 20 45 65 648 12

30.7 Mean 2.3 StandardError 30.8 Median #N/A Mode 7.9 StandardDeviation 62 Variance Kurtosis -1.8 0.0 Skewness 21.5 Range 20.0 Minimum 41.5 Maximum 368 Sum 12 1Count 1

-1.1 0.3 455 381 836 6792 12

1

250

Table D. 2 Descriptive statistics for urinary aMT6s levels by gerbils at 9 weeks; including 2 gerbils whose day I levels of aMT6s were used in analyses. aMT6s ng/24-h Females

Mean Standard Error Median Mode Standard Deviation Variance Kurtosis Skewness Range Minimum Maximum Sum Count Males

Mean Standard Error Median Mode Standard Deviation Variance Kurtosis Skewness Range Minimum Maximum Sum lCount

aMT6s pglg BW/24-h

9w LF FEMALES

9W HF FEMALES

25.7 1.8 25.1 #N/A 6.4 41.0 0.3 -0.5 22.2 12.3 34.4 309 12 9W LF MALES

27.9 2.2 25.8 #N/A 7.7 58.8 -0.6 0.6 24.4 18.4 42.8 335 1 12 1

9W LF FEMALES

9W HF FEMALES

20.2 1.8 18.5 #N/A 6.2 38.3 -1.1 0.5 17.9 12.0 29.9 242 12

476 31 459 #N/A 109 11853 1.2 -0.7 411 233 644 5708 12

365 31 340 #N/A 106 11214 -1.0 0.6 310 242 552 4381 12

9W HF MALES

9W LF MALES

9W HF MALES

19.6 1.4 19.3 15.7 4.7 22.4 1.4 1.1 17.3 13.3 30.6 235 12 1

425 32.6 410 #N/A 113 12785 -0.7 0.4 376 252 628 5099 12 1

body weight 9 9W LF FEMALES

9w HF FEMALES

54 1.0 53 52 3.4 11.6 -0.8 0.6 10 so 60 648 12

55 1.1 55 54 4.0 16 -0.3 -0.1 13 48 61 662 12

9W LF MALES

9W HF MALES

320 66 21.5 1.4 299 65 #N/A 63.8 75 4.9 5568 24.1 0.3 -0.4 0.8 0.7 263 15 213 60 476 75 3841 790 12 1 12

61 1.0 61 60 3.5 12.4 -0.6 -0.1 12 55 67 736 1 12

251

TableD.3 Descriptive statistics for the urinary aMT6s levels excreted in 24-h by gerbils aged II Y2weeks(including 5 gerbils whose day 1 levels of aMT6s were used).

W

111/2 Females

Mean Standard Error Median Mode Standard Deviation Variance Kurtosis Skewness Range Minimum Maximum Sum Count

Mean Standard Error Median Mode Standard Deviation Variance Kurtosis Skewness Range Minimum Maximum Sum Count .

IIIAW

11 1/2 W

111/2

W

111/2

W

111/2W

LF FEMALES

HF FEMALES

LF FEMALES

HF FEMALES

LF FEMALES

HF FEMALES

27.7 2.0 26.3 #N/A 7.1 49.8 0.8 0.4 26.5 15.3 41.7 333 12

26.1 2.9 27.8 #N/A 9.5 90.2

467 29 478 #N/A 102 10340 0.2

407 40 413 #N/A 133 17612

59 1.7 57 64 5.9 35.1

-1.3 0.2 387 218 605 4473 11

-0.7 0.6 18 53 71 711 12

63 1.9 65 66 6.3 40 0.3

2W Males

body weight 9

aMT6s pgIg BW/24-h

aMT6s ng/24-h

-1.6 -0.1 26.3 13.1 39.4 287 11 ,/

2W

-0.2 369 272 642 5605 12 ,/

2W

P/2

W

I/

2W

-0.1 22 53 75 696 11 1 P/2

W

LF MALES

HF MALES

LF MALES

HF MALES

LF MALES

HF MALES

33.0 2.8 30.3 #N/A 9.8 95.4 1.3 1.1 35.2 20.2 55.4 396 12

21.9 1.6 22.9 #N/A 5.7 32.2

449 32 440 #N/A 112 12434

299 21 309 #N/A 74 5402

-0.5 -0.5 18.2 11.4 29.6 263 12

-0.7 0.2 356 288 644 5389 12

-0.8 -0.4 223 173 396 3584 12

73 2.0 71 71 6.9 47.2 0.1 0.6 24 62 86 878 12

73 1.1 72 71 3.8 14.4 0.1 0.2 14 66 80 876 12

252

Table DA Descriptivestatisticsfor the urinary aMT6s levels excretedin 24h by gerbils aged 16 weeks. aMT6s pgIg BW/24-h

aMT6s ng/24-h Females

16W LF FEMALES

16W HF FEMALES

16W LF FEMALES

16W HF FEMALES

16W LF FEMALES

16W HF FEMALES

29.7 2.4 31.2 #N/A 8.2 66.7

24.2 3.3 25.5 #N/A 11.6 134.4

442 36 458 #N/A 126 15981

346 42 375 #N/A 147 21604

67 1.6 67 4NIA 5.5 30.4

-0.2 -0.8 25.4 14.9 40.2 357 12

-1.4 -0.2 33.1 7.2 40.2 290 12

-0.2 -0.4 421 236 657 5305 12

-1.1 -0.1 457 115 572 4151 12

-1.0 0.6 16 61 77 810 12

68 2.9 69 69 9.9 99 2.8 0.4 42 49 91 815 12

Mean StandardError Median Mode StandardDeviation Variance Kurtosis Skewness Range Minimum Maximum Sum Count

16W LF MALES 34.1 4.2 32.2 #N/A 14.5 209.4

Males Mean StandardError Median Mode StandardDeviation Variance Kurtosis Skewness Range Minimum Maximum Sum lCount

body weight 9

1

-1.1 0.4 43.0 13.4 56.4 409 12

16W 16W 16W 16W 16W HF LF LF HF HF MALES MALES MALES MALES MALES 399 31.6 397 85 78 3.1 32.3 2.7 42.7 1.7 30.6 388 82 382 78 #N/A #N/A #N/A 81.1 #N/A 112 10.9 9.5 6.1 148 12487 89.4 118.8 21877 36.6 2.2 2.0 2.4 -0.7 -0.5 1.3 1.3 0.3 1.3 -0.2 394 38.6 34 472 19 278 19.3 181 73 68 671 57.8 108 652 87 4786 1014 379 4766 938 12 12 12 12 12 1 1 1 1 1

253

Table D. 5 Descriptive statisticsfor the urinary aMT6s levels excretedin 24-h by male gerbils aged9 weeks.It includesdata from: i) the longitudinal study; ii) additional gerbils on 2x 24-h urine collections. aMT6s ng/24-h MALES Mean Standard Error Median Mode Standard Deviation Variance Kurtosis Skewness Range Minimum Maximum Sum Count .

9W LF MALES

9W HF MALES

aMT6s pg/gBW/24-h 9W LF MALES

9W HF MALES

body weight 9 9W LF MALES

9W HF MALES

27.9 2.2 25.8 #N/A 7.7 58.7

21.3 1.1 20.2 15.7 5.1 26.2

425 33 410 #N/A 113 12778

348 19 346 #N/A 85 7163

66 1.4 65 62 4.9 23.8

61 1.0 62 64 4.6 22

-0.6 0.6 24.4 18.4 42.8 335 12

-0.4 0.5 18.3 13.3 31.6 426 20

-0.7 0.4 376 252 628 5099 12

-0.4 0.5 308 213 521 6955 20

-0.5 0.7 15 60 75 790 12

-0.7 -0.2 17 53 70 1227 20

254

Table D.6 Descriptivestatisticsfor the meantotal urinary aMT6slevelsexcreted by all gerbilsat II Y2weeks.It includesdatafrom: i) the longitudinalstudy;ii) additionalgerbilson 2x 24-h urine collections. aMT6s pg/g BW/24-h

aMT6s ng/24-h 11V2W

11Y2W

11Y2W

11 Y2W

body weight 9 11Y2W

11Y2W

FEMALES

LF FEMALES

HF FEMALES

LF FEMALES

HF FEMALES

LF FEMALES

HF FEMALES

Mean StandardError Median Mode StandardDeviation Variance Kurtosis Skewness Range Minimum Maximum Sum Count

26.6 1.2 26.2 26.3 6.7 44.5 0.4 0.3 29.5 12.2 41.7 797 30

24.4 1.5 24.0 #N/A 7.6 57.2

450 21 424 #N/A 117 13701 0.1 0.4 520 205 725 13505 30

389 23 369 #N/A 113 12781

59 0.9 58 64 4.7 22.5

63 1.5 63 66 7.2 52

-0.7 0.5 387 218 605 9344 24

-0.5 0.5 18 53 71 1779 30

-0.5 -0.2 28 48 76 1505 24

11Y2W

11Y2W

11Y2W

-1.0 0.3 26.3 13.1 39.4 587 24 11Y2W

11Y2W

11V2W

MALES

LF MALES

BF MALES

LF MALES

HF MALES

LF MALES

HIF MALES

Mean StandardError Median Mode StandardDeviation Variance Kurtosis Skewness Range Minimum Maximum Sum Count .

33.0 2.8 30.3 #N/A 9.8 95.4 1.3 1.1 35.2 20.2 55.4 396 12

21.7 1.4 20.9 #N/A 6.5 42.6

449 32 440 #N/A 112 12434 -0.7 0.2 356 288 644 5389 12

73 2.0 71 71 6.9 47.2 0.1 0.6 24 62 86 878 12

70 1.2 71 71 5.4 30

-0.4 0.5 24.0008 11.4 35.4008 456 21

310 19 291 #N/A 86 7404 0.5 0.7 342 173 515 6508 21

-0.3 -0.2 21 59 80 1465 21

255

Table D.7 Descriptivestatisticsfor urinary aMT6s levelsexcretedby gerbils aged16weeks.It includesdatafrom: i) longitudinal study; ii) additional gerbilson 2x 24-hoururine collections. aMT6s ngt24-h Females

Mean Error Standard Median Mode Deviation Standard Variance Kurtosis Skewness Range Minimum Maximum Sum Count Males

Mean StandardError Median Mode StandardDeviation Variance Kurtosis Skewness Range Minimum Maximum Sum Count

aMT6s pg/g BW/24-h

16W LF FEMALES

16W HF FEMALES

16W LF FEMALES

25.2 2.2 26.1 #NIA 9.5 89.8 -1.6 0.1 26.9 13.3 40.2 454 18

23.5 2.6 23.6 #N/A 10.4 109.1 -1.1 -0.1 33.0 7.2 40.2 377 16

380 33 364 #N/A 139 19228 -1.1 0.3 468 189 657 6846 18

16W LF

16W HF

MALES

16W HF FEMALES

body weight 9 16W LF FEMALES

16W HF FEMALES

340 34 376 #N/A 134 18016 -1.0 0.0 457 115 572 5437 16

66 1.4 66 68 6.1 37.3 0.2 -0.1 24 53 77 1190 18

68 2.3 69 69 9.1 83 2.5 0.5 42 49 91 1086 16

16W LF

16W HF

16W LF

16W HF

MALES

MALES

MALES

MALES

MALES

28.1 2.9 21.7 #N/A 13.1 172 0.3 1.2 43.0 13.4 56.4

30.4 2.0 29.7 #N/A 8.8 78 4.0 1.6 38.5 19.3 57.8

342 29 287 #N/A 132 17347 0.5 1.1 471 181 652

393 21 395 388 94 8900 2.9 1.2 409 263

80 2.0 79 81 9.0 82 3.1 1.4 41 67

7 1.4 77 71 6.4 41 -0.8 -0.1 22 65

590 21

607 20

7184 21

672 7854 20

108 1688 21

87 1537 20

256

Table D. 8 Descriptive statistics for mean urinary aMT6s levels excreted by includes in intervals. The 24-hour 48-hours 2x 28 table gerbils aged weeks; over 9 instanceswhen the levels of aMT6s in day 1 are used. aMT6s pg/gBW/24-h

aMT6s ng/24-h Females

bodyweight 9

28W 28W 28W 28W 28W 28W LF HF LF FIF HF LF FEMALES FEMALES FEMALES FEMALES FEMALES FEMALES

Mean

17.4

21.7

248

289

70

76

Standard Error

1.2

1.6

15

23

1.2

1.7

Median

17.5

18.7

249

248

70

76

#N/A

#N/A

#N/A

#N/A

67.9

#N/A

Standard Deviation

4.4

5.7

58

83

4.4

6.2

Variance

19.4

32.5

3332

6909

18.9

38

-0.1 1.0

0.1

-0.6 0.9

0.5

1.6

0.2

0.4

238

17

25

Mode

Kurtosis

-0.7

Skewness

17.6

-0.2 217

9.2

15.4

135

213

62

64

Maximum

23.9

33.0

353

450

79

89

Sum

244

282

3469

3759

983

983

14

13

14

13

14

13

28W

28W

28W

28W

28W

28W

LF MALES

HF MALES

LF MALES

HF MALES

LF MALES

HF MALES

24.4

27.8

273

306

95

91

1.6

2.6

34

30

6.0

2.5

Median

22.7

27.3

204

307

106

90

Mode

#N/A

#N/A

#N/A

#N/A

#N/A

#N/A

Standard Deviation

4.7

7.5

102

86

18.1

6.9

Variance

22.4

55.6

10407

7326

327.9

48

-2.2

13.3

247

-0.3 265

-0.3 42

-0.8 0.6

Range

-0.4 25.7

-2.2 0.3

-0.4

Skewness

-1.3 0.3

1.5

Minimum

18.5

13.9

166

162

72

83

Maximum

31.9

39.6

413

427

114

103

Sum

220

222

2461

2448

857

729

9

8

9

8

9

8

Range

-0.2 14.8

Minimum

Count Males

Mean Standard Error

Kurtosis

Count

20

257

Table E. I. Resultsof areasof ventral glands in gerbils 9 week low-F males I length breadth

area

sample

CIN

9WLFMI 9W LF M2 9W LF M3 9W LF M4 9W LF M5 9W LF M6 9W LF M7

2.45 2.3 2.0 2.0 1.8 1.8 2.55

cla

cm,

0.60 0.55 0.70 0.50 0.55 0.50 0.55

1.16 0.99 1.10 0.79 0.78 0.71 1.10 0.95 02

I mean SD

body weighi

(r)

67 62 63 61 58 54 67 61.7 47

9 week high-F males length I breadth

sample

CIO

9WHFMI 9W HF M2 9W 14FM3 9W HF M4 9W HF MS 9W RIFM6 9W HF M7 9W HF M8

2.4 2.6 2.3 2.55 2.15 2.3 2.4 1 2.5

28 week low-F males 28W LF MI 28W LF M2 28W LF M3 28W LF M4 28W LF M5 28W LF M6 28W LF M7 28W LF M8

2.6 2.7 2.3 2.4 2.2 2.2 2.5 1 1.9

0.8 0.7 0.7 0.6 0.6 0.6 0.6 0.5

28WHFMI 28W HF M2 28W HF M3 28W HF M4 28W HF M5 28W HF M6 28W HF M7 28W HF M8

sample

breadth

I

r.. .

COS

IIY: W LFFI 11Y2WLFF2 11YzWLFF3 11Y2WLFF4 11Y2WLFF5 11Y2WLFF6 11Y2WLFF7 11Y2WLFFS 11Y2WLFF9 11Y2WLFFIO 11YaWLFFI I II YW LFF12 11Y2WLFF13 llY2WLFF14 llY3WLFF15 11Y2WLFF16 IIY, WLFF17 llY3WLFF18 llY3WLFF19 11Y2WLFF20 11V2WLFF21 11Y2WLFF22 11Y2WLFF23 11 V2W LFF24

11YW LFF25

area

body weight

(2) 0.5 0.55 1.4 66 0.6 1.2 0.57 63 indistinguishable 65 indistinguishable 59 indistinguishable 55 indistinguishable 65 indistinguishable 61 indistinguishable 58 indistinguishable 56 indistinguishable 54 indistinguishable 53 indistinguishable 50 indistinguishable 50 indistinguishable 54 indistinguishable 51 indistinguishable 56 indistinguishable 56 indistinguishable 57 indistinguishable 60 indistinguishable 56 indistinguishable 54 indistinguishable 58 0.3 0.32 1.35 65 indistinguishable 54 0.9 0.3 0.21 1 59

1.6 1.6 1.5 1.3 1.5 1.9 1.55 1 1.5

0.45 0.45 0.5 0.45 0.5 0.45 0.6 0.45 MEAN STDEV

0.75 1.02 0.90 1.00 0.98 0.99 1.32 1.28 1.03 0.2

62 65 53 64 55 56 68 22 6 1 6"

0.6 0.5 0.6 0.7 0.55 0.8 0.7 0.75

1.13 83 0.82 86 1.25 97 1.37 103 0.99 85 1.76 93 1.46 95 1.47 1 87 1.28 91 0.3 70

11% week high-F females

28 week low-F females 28W LF Fl 28W LF F2 28W LF F3 28W LF F4 28W LF F5 28W LF F6 28W LF F7 28W LF F8

2.4 2.1 2.65 2.5 2.3 2.8 2.65 1 2.5

Imean SD

11% week low-F females length

0.4 0.5 0.5 0.5 0.6 0.55 0.7 065 I mean SD

weight

28 week high-F males

1.63 113 1.48 108 1.26 106 1.13 114 1.04 84 1.04 77 1.18 109 0.75 1 72 1.19 98 0.3 17.2

mean SD

CUB

body

0.57 73 0.57 71 0.59 72 0.46 70 0.59 69 0.67 72 0.73 79 0.53 1 75 0.59 1 72 .6 0.08 32

length

1 breadth 1

area cla Call cm, 1 indistinguishable indistinguishable 1.15 0.45 0.41 1.6 0.5 0.63 1.2 0.35 0.33 1.3 0.45 0.46 1.3 0.5 0.51 1.25 0.35 0.34 1.45 0.4 0.46 indistinguishable

sample

1 11Y2WHFFl 11YzWHFF2 11Y2WHFF3 11Y2XWIFP 11V2WHFF5 11Y2WHFF6 11V2WHFF7 11YsWHFF8 11Y2WHFF9 IIY2WHFFIO

body weight

fir) 58 55 63 76 63 67 71 70 68 61

28 weekhigh-F females 28W HF Fl 28W HF F2 28W HF F3 29W HF F4 28W HF F5 28W HF F6 28W HF F7 28W HF F8 28W HF F9 28W HF FIO 28W HF Ft 1 28WHFF12

1.60 0.90 1.70 1.60 1.75 1.70 1.60 1.6 1.5 1.5 1.6 1 1.6

0.50 0.40 0.50 0.40 0.55 0.40 0.50 0.5 0.45 0.5 0.5 0.45 J MEAN STDEV

0.63 73 0.28 64 0.67 78 0.50 78 0.76 74 0.53 68 0.63 72 0.63 73 0.53 78 0.59 73 0.63 78 0.57 1 89 0.58 1 74.8 0.12 6.2

258

Table E.2 Resultsof the t -testsassumingunequalvariancesbetweenthe body weights of gerbils at 7,9 and II Y2weeks I 7 weeks of age Mean Variance Observations MeanDifference Hypothesized df t Stat P(T