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Restricted Feeding Study in the Male and Female Pubertal Tier Assays

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Restricted Feeding Study in the Male and Female Pubertal Tier Assays Powered By Docstoc
					Assessing the Impact of Body
 Weight on Male and Female
   Pubertal Development


         EPA Special Study

             Tammy Stoker, PhD.
    Gamete and Early Embryo Biology Branch
       Reproductive Toxicology Division
              NHEERL, U.S. EPA
            Overview
 Present data from food restriction study
 Discuss results of previous studies
  using male and female pubertal
  protocols.
 Discuss examples of published data
  and conclusions evaluating the role of
  body weight and growth on pubertal
  events.
        Study Objectives
• Assess the relationship between growth
  rate and pubertal development
• Determine whether or not reduced body
  weights over a range of 2 – 20% confound
  the endpoints in the Female and Male
  Pubertal Protocols
            Methods
• Four groups of male and female
  Wistar rats (n=13) were fed 90%,
  80%, 70% and 60% of ad libitum
  controls
• Pecentages were based on 24 h food
  intake of controls.
      Female Pubertal Protocol

                          Postnatal Day
 3
 or       21         25            30               35                40
 4                                         VO
                Females: Dose, Examine for VO, Daily BW



Cull                               Required endpoints:
         Wean                                                                 Necropsy
pups                                  Growth
         Assign                       Age and weight at vaginal opening (VO)
                                      Vaginal cytology
         to Trt.                      Serum thyroxine and thyroid-stimulating hormones
                                      Ovarian and uterine weights and histology
                                      Liver, kidney, pituitary and adrenal weights
Body Weight: Female Wistar Rats
                       Body Weight at Necropsy
                        Female Pubertal Study
                      180

                      160

                      140
                                                                     *        Percent reduction in BW as
                                                                          *
Body Weight (grams)




                      120
                                                                                compared with control
                      100

                      80
                                                                              • Group 90 (2.0%)
                      60                                                      • Group 80 (4.6%)
                      40
                                                                              • Group 70 (12.1%)
                      20

                       0                                                      • Group 60 (18.8%)
                                Control   90        80          70       60

                                           % Dietary Modification


                            Mean +/- SEM (n=13): * P<0.05
 Reduction in BW on PND 33
               8.2% *
               15.2% *




22   24   26    28      30   32   34   36   38   40
                                                Age at VO
                                        40




        Age at Vaginal Opening (days)
                                        30




                                        20




                                        10




                                         0
                                               Control   90        80        70   60

                                                         % Dietary Modification




Group                                   Con      90%          80%          70%         60%

Mean                                    33       33.3         32.7         34.0        34.5
SEM                                     0.56     0.67         0.70         0.97        0.97
                                 Ovary Weight
                        0.07                                                     0.06


                        0.06




                                                                                        Ovary Weights (% of Body Weight)
                                                                                 0.05
Ovary Weights (Grams)




                        0.05
                                                                    *
                                                                    *            0.04

                        0.04
                                                                        **       0.03
                        0.03

                                                                                 0.02
                        0.02

                                                                                 0.01
                        0.01


                        0.00                                                     0.00
                                Control        90         80       70   60

                                               % Dietary Modification

                               Ovary Weights
                               Percent of Body Weight
                                                        * = p< .05 for absolute weight.
                                                        r = p< .05 for relative weight.
                                Uterus Weight
                         0.30                                                 0.18

                                                                              0.16




                                                                                     Uterus Weights (% of Body Weight)
                         0.25
                                                                              0.14
Uterus Weights (grams)




                         0.20                                                 0.12

                                                                              0.10
                         0.15
                                                                              0.08

                         0.10                                                 0.06

                                                                              0.04
                         0.05
                                                                              0.02

                         0.00                                                 0.00
                                 Control        90        80        70   60

                                                % Dietary Modification


                                 Uterus Weights
                                 Percent of Body Weight
 Thyroid Hormones: Female
        1.4                                                                                                                1.2


        1.2
                                                            TSH                                                            1.0                                           T3
        1.0




                                                                                                        Serum T3 (ng/ml)
                                                                                                                           0.8
        0.8
ng/ml




                                                                                                                           0.6
        0.6


        0.4                                                                                                                0.4


        0.2
                                                                                                                           0.2

        0.0
              Control   90        80                        70       60                                                    0.0
                        % Dietary Modification                                                                                        Control   90        80        70   60

                                                                                                                                                % Dietary Modification
                                                             60



                                                             50                                                                   T4
                                         Serum T4 (ng/ml)




                                                             40



                                                             30



                                                             20



                                                             10



                                                                 0
                                                                          Control   90        80        70                       60

                                                                                    % Dietary Modification
      Summary of Female
• Reduced body weight gains from 2.0 to
  18.8% (at necropsy)
  – no effect on VO, uterine weights or thyroid
    hormones
• Body weight decreases of 12.1 and 18.8%
  – decreased ovarian weight
• Importantly, there were no significant
  differences in any of the female
  reproductive endpoints at less than 12.1%
  decreased body weight.
Male Pubertal Protocol


 3
                           Postnatal Day
 or
 4      21       25   30      35             40           45                    50          53
                                                        PPS

             Males: Dose, Examine for PPS, Daily BW


Cull   Wean                     Required endpoints:
                                                                                        Male
                                   Growth
pups   Assign                       Age and weight at preputial separation (PPS)      Necropsy
                                    Serum thyroxine and thyroid-stimulating hormones
       to Trt.                     Thyroid Histology
                                   Seminal vesicle plus coagulating gland weight (with fluid)
                                    Ventral prostate weight, Levator ani/bulbocavernosus weight
                                    Testis and epididymal weights and histology
                                    Liver, kidney, adrenal and pituitary weights
Body Weight: Male Wistar Rats
Body Weight at Necropsy:
Male Wistar Rats

Percent reduction in BW as
   compared with control
    • Group 90 (2.2%)
    • Group 80 (4.4%)
   • Group 70 (12.5%)
   • Group 60 (20.7%)
Reduction in BW on PND 44

           8.5% *




                            19.0% *
Age at Preputial Separation
                           50



    Age (Postnatal days)
                           45


                           40


                           35


                           30
                                        % Dietary Modification



    Group Con                            90%     80%      70%    60%


    Mean                        44.3     44.4    45.2     43.5   44.7
    SEM                          0.55    0.62    0.57     0.75   0.61
Ventral Prostate Weight
             0.35                                                            0.004


             0.30

                                                                             0.003
             0.25




                                                                                     Percent of Body Weight
                                                                       *
Weight (g)




             0.20
                                                                             0.002
             0.15


             0.10
                                                                             0.001

             0.05


             0.00                                                            0.000
                              Control         90%      80%       70%   60%

                    Ventral Prostate Weight
                                              % Dietary Modification
                    Percent of Body Weight

                                               * = p< .05 for absolute weight.
                                               r = p< .05 for relative weight.
Seminal Vesicle Weight
             0.8                                                           0.25




                                                                           0.20
             0.6




                                                                                  Percent of Body Weight
Weight (g)




                                                                           0.15

             0.4                                                       *
                                                                           0.10


             0.2
                                                                           0.05




             0.0                                                           0.00
                             Control        90%      80%       70%   60%

                                            % Dietary Modification
                   Seminal Vesicle Weight
                   Percent of Body Weight
        Epididymis Weight
             0.30                                                           0.10



             0.25
                                                                      r*
                                                                            0.08




                                                                                   Percent of Body Weight
             0.20
Weight (g)




                                                                            0.06

             0.15

                                                                            0.04
             0.10


                                                                            0.02
             0.05



             0.00                                                           0.00
                              Control        90%      80%       70%   60%

                                             % Dietary Modification
                    Epi Weight
                    Percent of Body Weight
                         Testes Weight
             1.6                                                                0.7


             1.4                                                                0.6

             1.2                                                           r
                                                                      r         0.5




                                                                                      Percent of Body Weight
             1.0
Weight (g)




                                                                                0.4
             0.8
                                                                                0.3
             0.6

                                                                                0.2
             0.4


             0.2                                                                0.1


             0.0                                                                0.0
                            Control         90%       80%        70%      60%

                   Left Testis Weight        % Dietary Modification
                   Percent of Body Weight
          Thyroid Hormones-Male
                   60                                                                                              1.2



                   50                                                           T4                                 1.0
                                                                                                                                                                  T3
                                                                                                                                                            *     *
                                                                      *




                                                                                                Serum T3 (ng/ml)
Serum T4 (ng/ml)




                   40                                                                                              0.8

                                                                                    *
                   30                                                                                              0.6



                   20                                                                                              0.4



                   10                                                                                              0.2



                    0                                                                                              0.0
                        Control   90                          80     70         60                                       Control    90           80          70   60

                                   % Dietary Modification                                                                               % Dietary Modification




                                                               1.8

                                                               1.6
                                                                                                                                   TSH
                                                               1.4


                                                                                                                                   *
                                          Serum TSH (ng/ml)




                                                               1.2

                                                               1.0

                                                               0.8

                                                               0.6

                                                               0.4

                                                               0.2

                                                               0.0
                                                                          Control       90                         80    70        60

                                                                                         % Dietary Modification
           Male Summary
• Reductions in body weight gain from 2.2
  to 20.7% had
  – no effect on PPS or testes weight
• Decreases in body weight from 12.5 to
  20.7%
  – decreased T3 and T4
• Decreases in body weight of 20.7%
  – decreased ventral prostate, seminal vesicle
    and epididymal weights
• Importantly, there were no significant
  alterations of the male reproductive
  endpoints at decreases of less than 12.5%.
                                                            Adrenal Weights
                                                      Male                                                                                                                    Female
                     0.07                                                             0.025                                                         50                                                       0.04


                     0.06
                                                                                      0.020                                                         40
                                                                                                                                                                                                    *    *




                                                                                                                       Paired Adrenal Weight (mg)
                                                                                                                                                                                                             0.03
                     0.05




                                                                                                                                                                                                                    Adrenal (% body weight)
                                                                                              Percent of Body Weight
Weight (pair in g)




                                                                                      0.015                                                         30
                     0.04
                                                     *                     *
                     0.03
                                                                                *     0.010                                                         20
                                                                                                                                                                                                             0.02




                     0.02
                                                                                                                                                                                                             0.01
                                                                                      0.005                                                         10
                     0.01


                     0.00                                                             0.000                                                         0                                                        0.00
                                      Control        90%       80%        70%   60%                                                                        control       90         80          70      60

                            Adrenal Weight            % Dietary Modification                                                                                               % Dietary Modification
                            Percent of Body Weight

                                                                                                                                                         Adrenal Weight
                                                                                                                                                         Percent of Body Weight




                                                               * = p< .05 for absolute weight.
                                                               r = p< .05 for relative weight.
                    Anterior Pituitary Weight

                                               Male                                                                               Female
              12                                                          0.004                            10                                                    0.006



              10                                                                                                                                                 0.005
                                                                                                           8
                                                                                                                                                          * *




                                                                                                                                                                         Pituitary (% of Body Weight)
                                                                          0.003

                                                   *


                                                                                  Pituitary Weight (mg)
                                                                                  Percent of Body Weight
               8
                                                               *                                                                                                 0.004
Weight (mg)




               6
                                                                     *    0.002
                                                                                                           6

                                                                                                                                                                 0.003

                                                                                                           4
               4                                                                                                                                                 0.002

                                                                          0.001
                                                                                                           2
               2                                                                                                                                                 0.001



               0                                                          0.000                            0                                                     0.000
                            control       90        80          70   60                                          Control        90         80         70    60

                                           % Dietary Modification                                                                % Dietary Modification
              Anterior Pituitary Weight
              Percent of Body Weight
                                                                                                                Pituitary Weight
                                                                                                                Percent of Body Weight
                                                              Kidney Weights

                                                     Males                                                                                                      Females
                     3.0                                                             1.0                                                    1.8


                                                                                r                                                           1.6
                                                                                                                                                                                                          1.0




                                                                                                                                                                                                                Kidney Weight (% of Body Weight)
                     2.5
                                                                                     0.8                                                    1.4
                                                                                                                                                                                             *        *   0.8




                                                                                                                    Kidney Weight (grams)
                                                              *
                                                                                           Percent of Body Weight
                                                                                                                                            1.2
Weight (pair in g)




                     2.0

                                                                         *           0.6

                     1.5                                                       *                                                            1.0

                                                                                                                                            0.8
                                                                                                                                                                                                          0.6



                                                                                     0.4
                                                                                                                                                                                                          0.4
                     1.0                                                                                                                    0.6

                                                                                                                                            0.4
                                                                                     0.2                                                                                                                  0.2
                     0.5
                                                                                                                                            0.2


                     0.0                                                             0.0                                                    0.0                                                           0.0
                                    Control         90%       80%        70%   60%                                                                 Control        90        80          70       60

                                                     % Dietary Modification                                                                                        % Dietary Modification
                           Kidney Pair Weight
                           Percent of Body Weight
                                                                                                                                                  Kidney weight
                                                                                                                                                  Percent of Body Weight
                                                                 Liver Weight
                                           Males                                                                                                 Females
                                                                                                                                8                                                       6
             16

                                                      r r                   5



                                                                      r                                                                                                                 5




                                                                                                                                                                                            Liver Weight (% of Body Weight)
             14


             12
                                                                            4                                                   6
                                                                                                                                                                               *   r


                                                                                                         Liver Weight (grams)
                                           * *                                                                                                                                     *    4




                                                                                Percent of Body Weight
             10

                                                                *
Weight (g)




                                                                            3

                                                                                                                                4                                                       3
             8


             6
                                                                      *     2
                                                                                                                                                                                        2
             4                                                                                                                  2
                                                                            1
                                                                                                                                                                                        1
             2


             0                                                              0
                                                                                                                                0                                                       0
                          Control          90%       80%        70%   60%
                                                                                                                                      Control        90         80         70      60
                                            % Dietary Modification
                  Liver Weight                                                                                                                        % Dietary Modification
                  Percent of Body Weight

                                                                                                                                    Liver Weight
                                                                                                                                    Percent of Body Weight
      Study Conclusions
• A 10% reduction in body weight has
  no effect on pubertal development
• Pubertal protocols detect a wide
  variety of EDCs apart from modest
  decreases in BW
       Background Literature

• Studies were designed to retard
  reproductive senescence and
  prolong lifespan:
   Litter alterations during early postnatal days resulted in
    30 - 65% bw difference.
   Other studies severely restricted food intake at weaning.
   Indeed, these studies found that puberty was delayed.
   The first week of life is critical for nourishment and
    setting of adult bw.
Continued:
•   “attainment of critical bw” hypothesis
•   “critical body fat” hypothesis
•   “growth rate” hypothesis
•   All have been questioned by more recent
    investigations which were unable to repeat earlier
    studies(Aguilar et al.,1984; Glass et al., 1984,
    Ronnekleiv, Ojeda & McCann, 1978;Bronson,
    2001; Crawford and Osler, 1975).
General agreement in literature
• A 10% decrease in body weight gain in the
  pubertal assays does not appear to
  confound the selected endpoints (Connor
  et al., 2000).
• The reproductive systems in the adult
  male and female rat are relatively resistant
  to body weight reductions down to 70% of
  control (Chapin et al., 1993).
Relationship between reduction in body
 weight versus change in age at PPS
                              Feeding       Linuron   DDE2      DBP2         MT-TH
                              Vinclozolin   DDE1      DBP       KETO-TH      FLUT-TH

                        15
Change in PPS in Days




                        10

                         5

                         0

                         -5

                        -10
                                 100         95           90          85         80
                                            Percent of control body weight
Relationship between reduction in
 body weight versus age at PPS
                              Feeding        DACT 100
                                AT R         DACT 200
                          5
                        4.5
                          4
Delay in PPS in Days




                        3.5
                                                                               200
                          3
                        2.5                    12.5
                          2             50            100   150
                        1.5
                          1
                        0.5
                          0
                       -0.5
                         -1
                       -1.5
                                 100           95             90         85          80
                                              Percent of control body weight
Relationship Between Body Weight and Age
            at Vaginal Opening


                                15
  Change in Age at VO ( Days)




                                10

                                                                        ATR200
                                 5                   ATR100
                                        ATR50
                                                                                              Feeding
                                 0


                                 -5

                                            MXC100        EE.005                      TAM10
                                -10
                                      100            95            90            85              80
                                                 Percent of control body weight
Does the estrogen mediated suppression of
  appetite confound the detection of VO?

                                0
                                 1                Female VO
      VO Acceleration in Days

                                2
                                 3
                                4
                                5
                                6
                                7
                                8
                                9
                                10
                                      100   98    96   94    92   90       88
                                     Percent of Control Weight at day 42

                 Methoxychlor (0, 25, 50, 100, 200 mg/kg)
Acknowledgments
• Collaborators               • Technical Assistance
  –   Susan Laws, Ph.D.           –   Janet Ferrell
  –   Ralph Cooper, Ph.D.         –   Keith McElroy
  –   Earl Gray, Ph.D.            –   Kate Bremser
  –   Jerome Goldman, Ph.D.       –   Korin Elliott
  –   Robert Kavlock, Ph.D.


              Reproductive Toxicology Division
                  NHEERL, ORD, U.S. EPA

				
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