Life Sciences Vol . 21, pp . 63-70 Printed in the U.S .A .
Pergamon Press
THE EFFECTS OF PROLONGED COFFEE INTAKE ON GENETICALLY IDENTICAL MICE A. Robert Bauer, Jr ., Robert K. Rank, Roger Rerr Robert L . Straley and J. David Mason* Research Laboratory, Department of Pathology Central Michigan Community Hospital Mt . Pleasant, Michigan *Department of Mathematics, University of Utah Salt Lake City, Utah (Received in final form May 26, 1977) SUMMARY C57BL6J mice were fed coffee over a prolonged period of time (adult life) . They had a statistically significant decrease in longevity as compared to the genetically identical controls . The coffee fed animals developed a markedly deteriorated physical condition and lower weights despite the fact that they consumed as much or more food and fluid than the water fed controls . Coffee has been widely accepted thoughout the world as a beverage and a stimulant . Although there have been questions regarding its toxicity from the time of its first use, this acceptance has nevertheless been based on the assumption that coffee caused little harm and may even be of some benefit to adult man in the performance of his daily activities (1,2) . Despite the fact that considerable work has been done on the effects of various caffeine mixtures on toxicity, growth, reproduction, mutagenic and possible carcinogenic effects (3,4,5,6,7,8,9,10,11,12), as well as inconclusive statistical population studies on a possible relationship of coffee drinking to bladder cancer (13,14,15), there is a lack of well controlled long term studies on the effect of brewed coffee, as it is commonly consumed in North America. It is difficult to establish a controlled study of coffee intake in man since a considerable number of hereditary and environmental differences can influence the results . In recent years genetically identical strains of mice have become available for various research endeavors (16) . It is apparent that these essentially identical mammals can be used for evaluating the effects of various foods and environmental factors on longevity, nutrition, appearance and various physiological processes . These evaluations, under controlled conditions, may help provide a clearer answer to the effect of coffee on man. It was the purpose of this experiment to measure the effects of coffee as prepared and drunk by many people in North America on the longevity of pure-bred strain mice of the same age and sex. These coffee test mice were kept under conditions identical to their randomly selected litter mates . From the time of an estimated early adulthood until death, the test animals drank coffee instead of water as the only variable . 63
64
Prolonged Coffee Intake
Vol . 21, No . 1, 1977
METHOD A total of 109 C57BL6J strain mice were used in this experiment . These mice, all of male gender, were obtained from the Jackson Laboratory*. The life span of three different groups were studied in experiments conducted from April, 1966, until May, 1973 . Group 1 . The first experimental group, a pilot group, consisted of three coffee test animals and three control animals. Genetically identical litter mates were randomly selected . Their weights were recorded throughout the remainder of their lifetime from the start of the experiment in April, 1966 . In the first experiment the coffee was started when the mice were approximately 2 months of age . They had an estimated life span of 24+ months (17) . The coffee and water were changed each day and the animals were weighed weekly, with recorded observations of appearance, activity and eating habits . Freshly brewed coffee without additives was offered as the sole available fluid to the coffee test mice throughout the experiment . Group 2 . In the second group of test animals, consisting of 50 mice, 25 animals were used for the coffee test group and 25 animals were used for the water control group . These animals were randomly selected for each group . They were born in December, 1967 and were started on coffee in July, 1968 . The mice in the second group were studied primarily for longevity and for pathological tissue changes. Group 3 . The third experimental group was started in February, 1971 . These 53 mice were born in October, 1970, and were divided with random aelection into 27 animals in the teat group and 26 animals in the control group . They were studied for quantity of fluid and food intake as well as longevity . In experimental Groups 2 and 3 fresh coffee was brewed each day under standard conditions . A similar quantity of water was boiled under identical conditions . The coffee and boiled water were allowed to cool to room temper ature prior to being made available to the animals. The animals were kept in clear plastic cages with wire tops and no more than 5 animals to a cage . They were fed standard rodent chow** . There was always sufficient excess food in the cage to allow the animals all the food they desired at any time . There was also always more coffee and more water Standard commerical regular in the animals' cages than they could consume . grind coffee was used (5 .2 gm/100cc water) . The brewing time was controlled by automatic timers . In addition, an exactly similar experiment was conducted at the same time with AJax strain mice obtained also from the Jackson Laboratory . These were albino mice of a strong genetically different (H2 locus) strain than the black C57BL6J mice . RESULTS C57BL6J strain mice were, as outlined above, divided into 3 groups in this experiment . There were 109 animals with 55 coffee animals and 54 control animals . C57 coffee mice lived consistently shorter lives than the C57 control animals . This is statistically documented by the Nathan Mantel teat (18, see addendum) . * The Jackson Laboratory, Bar Harbor, Maine ** Purina Chow
Vol . 21, No . 1, 1977
Prolonged Coffee Intake
65
Group 1 . In Group 1, it was noted that although the animals started out with similar weights, the coffee fed mice subsequently had lower weights even though they ate more food and appeared to drink as much or more fluid than the control animals . It was also noted that the animals drinking coffee generally appeared to age sooner (Illustration 1) . They had a lose of fur and developed It was also noted that very shaggy coats as compared to the control animals . the coffee test animals often developed changes in their tails and lost portions of their tails (Illustration 2) . In this pilot experiment the 3 coffee animals died much sooner than the 3 control animals (Graph 5) . In the Group 2 C57BL6J animals it was noted that coffee animals Group 2 . died sooner than control animals (P ~ 0 .009, see addendum) . The gradual pattern of mortality of the coffee test animals as compared to controls can be Postmortem examination observed on the longevity curves illustrated is Graph 1 . did not demonstrate any lesions or disease pattern that could be considered exclusively responsible for the coffee test animals' deaths . The examination included gross and microscopic study of the lungs, heart, great vessels, esophagus, diaphragm, liver, spleen, pancreas, stomach, intestines, colon, No unusual occurance of tumors or kidney, bladder, skeletal muscle and akin . cancers was found in the teat animals . Pneumonia was often found as a terminal event in both the test and control mice . An electron microscopic study of a constant portion of the aorta by one of the authors (RK) did not demonstrate any pattern or Vascular lesion in the test animals .
rLLUSTRATION 1 C57BL6J (Group 2) coffee fed mice (left) as compared to water fed controls (right) . Note absence of hair, changes in tail and general poorer nutritional status .
66
Prolonged Coffee Intake
Vol . 21, No . 1,
1977
ILLUSTRATION 2 C57BL6J (Group 1) coffee fed mouse (above) as compared to water fed control . Note changes in tail of coffee fed mouse, kept in separate cage . 25-i~
_
_ __
_
_ _
___
__________
dROUP2 C51 BL 8J
_______1,
~a
` ~~l_
E 15'
COFFEE CONTROL-_.
= 105-
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ss
i
rs
LorgevityExpeAment Time (YJed~e)
~oo
GRAPH 1 Longevity curve of the Group 2 C57BL6J coffee teat mice (lower curve) compared to their identical water fed controls (upper curve) . The coffee fed mice died sooner (P ~ 0 .009) .
Vol. 21, No . 1,
1977
Prolonged Coffee Intake
67
Group 3 . In Group 3, the C57 coffee teat group again demonstrated leas longevity and a gradual pattern of mortality (Graph 2) (P ~ 0.015, see addendum) . The measured intake of fluid and food throughout this experiment consistently showed a higher quantity intake of fluid and a consumption of as much or more food by the coffee fed animals (Graphs 3 and 4) . These findings were also observed in Group 1 and 2 animals .
GRAPH 2 Longevity curve of the Group 3 C57BL6J coffee test mice (lower curve) compared to their water fed controls (upper curve) . The coffee fed mice died much sooner (P ~ 0 .015) . GROUP3 C57 FLUID INTAKE COFFEE CONTROL.. .. . . .. . ..
GRAPH 3 Illustrative portion of plotted daily fluid intake of coffee fed Group 3 C57BL6J mice (cc/mouse/day) (upper line) as compared to water controls (lower line) . The coffee test animals generally consumed as much or more fluid than the controls .
GRAPH 4 The average daily food intake of Group 3 C57BL6J coffee teat mice (upper line) as compared to genetically identical randomly selected water controls (lower line) . The food intake of the coffee fed mice (gm/mouse/day) was generally as much or more than the food intake of the water fed controls until they were terminal .
95
50 E~srtyrrtTm~-IW~e4i1
75
GRAPR 5 Weights of Group 1 C57BL6J coffee fed mice as compared to their water fed controls . The coffee fed mice consistently weighed less although they consumed as much or more fluid and food than the controls .
Vol. 21, No . 1,
1977
Prolonged Coffee Intake
69
Group 1 and Group 3 animals from the AJax strain demonstrated similar findings to those found with the C57 strain mice . The Group 2 AJax mice had early frequent lymphomas develop in the control and teat groups destroying any mortality pattern . Both AJax Group 1, a pilot group, and Group 3 mice had early coffee test deaths as compared to their controls (Group 3, P - 0 .046) . The coffee teat animals also had increases in fluid and food intake and decreases in weight similar to the C57 mice . No consistent pattern of pathological lesion or disease different from controls was noted among the AJax coffee test animals on a thorough gross and microscopic postmortem examination of body tissue . DISCUSSION It was not the purpose of thin experiment to determine the ingredients in coffee which could cause harm, but to establish whether coffee in fact did cause changes in a mammal with constant use over a prolonged period of time . The results demonstrate that brewed coffee fed over a prolonged period of time was detrimental to the longevity of C57BL6J mice as compared to genetically identical water fed controls . The coffee test animals demonstrated a markedly deteriorated physical condition and lower weights even though they drank and ate ae much or more fluid and food than their controls . It should be mentioned .that Strubelt et al also noted a decreased weight in shorter-term coffee fed rata (19) . Similar physical changes were noted in albino rats on high doses of caffeine by Boyd et al and Pfeiffer (5,9) perhaps on a "psychotic" basis . Ruhlmann et al (9) noted premature aging in the Drosophila due to Perhaps premature caffeine induced chromosome breakage with somatic damage . aging is responsible for the changes noted in the coffee fed mice in this study. Biochemical studies were not done during this experiment and will probably give a more definite answer to the coffee effect in the future . The same findings were noted in the AJax strain mice, an albino mouse with a strong H2 locus difference from the black C57BL6J mice, indicating that the coffee effect is not limited to one genetic strain of mice but perhaps of a more general nature . ADDENDUM An extension to the Mantel-Haenazel procedure was used to teat the significance of the data . This procedure is discussed in detail in "Evaluation of Survival Data and Two New Rank Order Statistics Arising in its Consideration" by Nathan Mantel in Cancer Chemotherapy Reports Vol . 50, No . 3, March, 1966, pages 163-170. Briefly, the procedure consists of dividing the data into several 2x2 tablea over nonoverlapping time periods, analyzing each table with the usual chi-square method, then combining the expected values and variances into one chi-square . The following table was obtained . chi-square C57 Group II C57 Group III AJax Group III
6 .97 6 .035 4 .014
*Department of Mathematics University of Utah, Salt Lake City, Utah
significant probability .009 .015 .046
J . David Mason, Ph .D .
70
Prolonged Coffee Intake
Vol . 21, No . 1, 1977
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 .
Nutr . Rev . _28 38-40 (1970) . J . M . RITCHIE, Central Nervous System Stimulants (Chap . 19) L . S . GOODMAN and A . GILMAN, The Pharmacological Basis of Theraputics, 5th _Ed . p . 376, MacMillian Pub . Co ., N .Y . (1975) . P . S . THAYER and C . J . RENSLER, Toxicol . Appl . Pharmacol . _25 169-179 (1973) . G . BACHMAN, J . HALDI, W . WYNN, and C . ENSOR, J . Nutr . _32 239-247 (1946) . E . M . BOYD, M . DOLMAN, L . M . KNIGHT, and E . P . SHEPPARD, Canad . J . Phyaiol . Pharmacol . _43 995-1007 (1965) . C . C . SCOTT, R . C . ANDERSON, and K . K . CHEN, J . Pharmacol . Exptl . Therap . _86 113-119 (1946) . E . E . FOLTZ, A . C . IVY, and C . J . BARBORKA, J . Lab . Clin . Med . _28 603-606 (1943) . C . J . PFEIFFER and G . H . GASS, Canad . J . Biochem . Phyaiol . _40 1473-1476 (1962) . W . KUIiLMANN, H . G . FROMME, E . M . HEEGE, and W . 05TERTAG, Cancer Res . _28 2375-2383 (1968) . S . EPSTEIN and H . SHAFNER, Nature _219 385-387 (1968) . M . KURATSUNE and W . C . HUEPER, J . Nat . Cancer Inst . _20 37-51 (1958) . P . J . DONOVAN and J . A . DIPAOLO, Cancer Res . 34 2720-2727 (1974) . J . SIMON, S . YEN,'and P . COLE, J . Nat . CancerÎnat . _54 587-591 (1975) . R . W . MORGAN and M . G . JAW , Can . Med . Asaoc . J . _111 1067-1070 (1974) . I . D . J . BROS S and J . TIDINGS, Prev . Med . _2 445-451 (1973) . E . L . GREEN, Am . Zoll . _3 358-360 (1963) . E . L . GREEN, Handbook on Genetically Standardized Jax Mice , p .3 Bar Harbor Pub . Co ., Bar Harbor, Maine (1962) . N . MANTEL, Cancer Chemother . Rep . _50 163-170 (1966) . 0 . STAUBELT, C . P . SIEGERS, H . BREWING, and J . STEFFEN, Z . Ernaehrungswias 12 252-260 (1973) .
Pergamon Press
THE EFFECTS OF PROLONGED COFFEE INTAKE ON GENETICALLY IDENTICAL MICE A. Robert Bauer, Jr ., Robert K. Rank, Roger Rerr Robert L . Straley and J. David Mason* Research Laboratory, Department of Pathology Central Michigan Community Hospital Mt . Pleasant, Michigan *Department of Mathematics, University of Utah Salt Lake City, Utah (Received in final form May 26, 1977) SUMMARY C57BL6J mice were fed coffee over a prolonged period of time (adult life) . They had a statistically significant decrease in longevity as compared to the genetically identical controls . The coffee fed animals developed a markedly deteriorated physical condition and lower weights despite the fact that they consumed as much or more food and fluid than the water fed controls . Coffee has been widely accepted thoughout the world as a beverage and a stimulant . Although there have been questions regarding its toxicity from the time of its first use, this acceptance has nevertheless been based on the assumption that coffee caused little harm and may even be of some benefit to adult man in the performance of his daily activities (1,2) . Despite the fact that considerable work has been done on the effects of various caffeine mixtures on toxicity, growth, reproduction, mutagenic and possible carcinogenic effects (3,4,5,6,7,8,9,10,11,12), as well as inconclusive statistical population studies on a possible relationship of coffee drinking to bladder cancer (13,14,15), there is a lack of well controlled long term studies on the effect of brewed coffee, as it is commonly consumed in North America. It is difficult to establish a controlled study of coffee intake in man since a considerable number of hereditary and environmental differences can influence the results . In recent years genetically identical strains of mice have become available for various research endeavors (16) . It is apparent that these essentially identical mammals can be used for evaluating the effects of various foods and environmental factors on longevity, nutrition, appearance and various physiological processes . These evaluations, under controlled conditions, may help provide a clearer answer to the effect of coffee on man. It was the purpose of this experiment to measure the effects of coffee as prepared and drunk by many people in North America on the longevity of pure-bred strain mice of the same age and sex. These coffee test mice were kept under conditions identical to their randomly selected litter mates . From the time of an estimated early adulthood until death, the test animals drank coffee instead of water as the only variable . 63
64
Prolonged Coffee Intake
Vol . 21, No . 1, 1977
METHOD A total of 109 C57BL6J strain mice were used in this experiment . These mice, all of male gender, were obtained from the Jackson Laboratory*. The life span of three different groups were studied in experiments conducted from April, 1966, until May, 1973 . Group 1 . The first experimental group, a pilot group, consisted of three coffee test animals and three control animals. Genetically identical litter mates were randomly selected . Their weights were recorded throughout the remainder of their lifetime from the start of the experiment in April, 1966 . In the first experiment the coffee was started when the mice were approximately 2 months of age . They had an estimated life span of 24+ months (17) . The coffee and water were changed each day and the animals were weighed weekly, with recorded observations of appearance, activity and eating habits . Freshly brewed coffee without additives was offered as the sole available fluid to the coffee test mice throughout the experiment . Group 2 . In the second group of test animals, consisting of 50 mice, 25 animals were used for the coffee test group and 25 animals were used for the water control group . These animals were randomly selected for each group . They were born in December, 1967 and were started on coffee in July, 1968 . The mice in the second group were studied primarily for longevity and for pathological tissue changes. Group 3 . The third experimental group was started in February, 1971 . These 53 mice were born in October, 1970, and were divided with random aelection into 27 animals in the teat group and 26 animals in the control group . They were studied for quantity of fluid and food intake as well as longevity . In experimental Groups 2 and 3 fresh coffee was brewed each day under standard conditions . A similar quantity of water was boiled under identical conditions . The coffee and boiled water were allowed to cool to room temper ature prior to being made available to the animals. The animals were kept in clear plastic cages with wire tops and no more than 5 animals to a cage . They were fed standard rodent chow** . There was always sufficient excess food in the cage to allow the animals all the food they desired at any time . There was also always more coffee and more water Standard commerical regular in the animals' cages than they could consume . grind coffee was used (5 .2 gm/100cc water) . The brewing time was controlled by automatic timers . In addition, an exactly similar experiment was conducted at the same time with AJax strain mice obtained also from the Jackson Laboratory . These were albino mice of a strong genetically different (H2 locus) strain than the black C57BL6J mice . RESULTS C57BL6J strain mice were, as outlined above, divided into 3 groups in this experiment . There were 109 animals with 55 coffee animals and 54 control animals . C57 coffee mice lived consistently shorter lives than the C57 control animals . This is statistically documented by the Nathan Mantel teat (18, see addendum) . * The Jackson Laboratory, Bar Harbor, Maine ** Purina Chow
Vol . 21, No . 1, 1977
Prolonged Coffee Intake
65
Group 1 . In Group 1, it was noted that although the animals started out with similar weights, the coffee fed mice subsequently had lower weights even though they ate more food and appeared to drink as much or more fluid than the control animals . It was also noted that the animals drinking coffee generally appeared to age sooner (Illustration 1) . They had a lose of fur and developed It was also noted that very shaggy coats as compared to the control animals . the coffee test animals often developed changes in their tails and lost portions of their tails (Illustration 2) . In this pilot experiment the 3 coffee animals died much sooner than the 3 control animals (Graph 5) . In the Group 2 C57BL6J animals it was noted that coffee animals Group 2 . died sooner than control animals (P ~ 0 .009, see addendum) . The gradual pattern of mortality of the coffee test animals as compared to controls can be Postmortem examination observed on the longevity curves illustrated is Graph 1 . did not demonstrate any lesions or disease pattern that could be considered exclusively responsible for the coffee test animals' deaths . The examination included gross and microscopic study of the lungs, heart, great vessels, esophagus, diaphragm, liver, spleen, pancreas, stomach, intestines, colon, No unusual occurance of tumors or kidney, bladder, skeletal muscle and akin . cancers was found in the teat animals . Pneumonia was often found as a terminal event in both the test and control mice . An electron microscopic study of a constant portion of the aorta by one of the authors (RK) did not demonstrate any pattern or Vascular lesion in the test animals .
rLLUSTRATION 1 C57BL6J (Group 2) coffee fed mice (left) as compared to water fed controls (right) . Note absence of hair, changes in tail and general poorer nutritional status .
66
Prolonged Coffee Intake
Vol . 21, No . 1,
1977
ILLUSTRATION 2 C57BL6J (Group 1) coffee fed mouse (above) as compared to water fed control . Note changes in tail of coffee fed mouse, kept in separate cage . 25-i~
_
_ __
_
_ _
___
__________
dROUP2 C51 BL 8J
_______1,
~a
` ~~l_
E 15'
COFFEE CONTROL-_.
= 105-
o-
ss
i
rs
LorgevityExpeAment Time (YJed~e)
~oo
GRAPH 1 Longevity curve of the Group 2 C57BL6J coffee teat mice (lower curve) compared to their identical water fed controls (upper curve) . The coffee fed mice died sooner (P ~ 0 .009) .
Vol. 21, No . 1,
1977
Prolonged Coffee Intake
67
Group 3 . In Group 3, the C57 coffee teat group again demonstrated leas longevity and a gradual pattern of mortality (Graph 2) (P ~ 0.015, see addendum) . The measured intake of fluid and food throughout this experiment consistently showed a higher quantity intake of fluid and a consumption of as much or more food by the coffee fed animals (Graphs 3 and 4) . These findings were also observed in Group 1 and 2 animals .
GRAPH 2 Longevity curve of the Group 3 C57BL6J coffee test mice (lower curve) compared to their water fed controls (upper curve) . The coffee fed mice died much sooner (P ~ 0 .015) . GROUP3 C57 FLUID INTAKE COFFEE CONTROL.. .. . . .. . ..
GRAPH 3 Illustrative portion of plotted daily fluid intake of coffee fed Group 3 C57BL6J mice (cc/mouse/day) (upper line) as compared to water controls (lower line) . The coffee test animals generally consumed as much or more fluid than the controls .
GRAPH 4 The average daily food intake of Group 3 C57BL6J coffee teat mice (upper line) as compared to genetically identical randomly selected water controls (lower line) . The food intake of the coffee fed mice (gm/mouse/day) was generally as much or more than the food intake of the water fed controls until they were terminal .
95
50 E~srtyrrtTm~-IW~e4i1
75
GRAPR 5 Weights of Group 1 C57BL6J coffee fed mice as compared to their water fed controls . The coffee fed mice consistently weighed less although they consumed as much or more fluid and food than the controls .
Vol. 21, No . 1,
1977
Prolonged Coffee Intake
69
Group 1 and Group 3 animals from the AJax strain demonstrated similar findings to those found with the C57 strain mice . The Group 2 AJax mice had early frequent lymphomas develop in the control and teat groups destroying any mortality pattern . Both AJax Group 1, a pilot group, and Group 3 mice had early coffee test deaths as compared to their controls (Group 3, P - 0 .046) . The coffee teat animals also had increases in fluid and food intake and decreases in weight similar to the C57 mice . No consistent pattern of pathological lesion or disease different from controls was noted among the AJax coffee test animals on a thorough gross and microscopic postmortem examination of body tissue . DISCUSSION It was not the purpose of thin experiment to determine the ingredients in coffee which could cause harm, but to establish whether coffee in fact did cause changes in a mammal with constant use over a prolonged period of time . The results demonstrate that brewed coffee fed over a prolonged period of time was detrimental to the longevity of C57BL6J mice as compared to genetically identical water fed controls . The coffee test animals demonstrated a markedly deteriorated physical condition and lower weights even though they drank and ate ae much or more fluid and food than their controls . It should be mentioned .that Strubelt et al also noted a decreased weight in shorter-term coffee fed rata (19) . Similar physical changes were noted in albino rats on high doses of caffeine by Boyd et al and Pfeiffer (5,9) perhaps on a "psychotic" basis . Ruhlmann et al (9) noted premature aging in the Drosophila due to Perhaps premature caffeine induced chromosome breakage with somatic damage . aging is responsible for the changes noted in the coffee fed mice in this study. Biochemical studies were not done during this experiment and will probably give a more definite answer to the coffee effect in the future . The same findings were noted in the AJax strain mice, an albino mouse with a strong H2 locus difference from the black C57BL6J mice, indicating that the coffee effect is not limited to one genetic strain of mice but perhaps of a more general nature . ADDENDUM An extension to the Mantel-Haenazel procedure was used to teat the significance of the data . This procedure is discussed in detail in "Evaluation of Survival Data and Two New Rank Order Statistics Arising in its Consideration" by Nathan Mantel in Cancer Chemotherapy Reports Vol . 50, No . 3, March, 1966, pages 163-170. Briefly, the procedure consists of dividing the data into several 2x2 tablea over nonoverlapping time periods, analyzing each table with the usual chi-square method, then combining the expected values and variances into one chi-square . The following table was obtained . chi-square C57 Group II C57 Group III AJax Group III
6 .97 6 .035 4 .014
*Department of Mathematics University of Utah, Salt Lake City, Utah
significant probability .009 .015 .046
J . David Mason, Ph .D .
70
Prolonged Coffee Intake
Vol . 21, No . 1, 1977
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 .
Nutr . Rev . _28 38-40 (1970) . J . M . RITCHIE, Central Nervous System Stimulants (Chap . 19) L . S . GOODMAN and A . GILMAN, The Pharmacological Basis of Theraputics, 5th _Ed . p . 376, MacMillian Pub . Co ., N .Y . (1975) . P . S . THAYER and C . J . RENSLER, Toxicol . Appl . Pharmacol . _25 169-179 (1973) . G . BACHMAN, J . HALDI, W . WYNN, and C . ENSOR, J . Nutr . _32 239-247 (1946) . E . M . BOYD, M . DOLMAN, L . M . KNIGHT, and E . P . SHEPPARD, Canad . J . Phyaiol . Pharmacol . _43 995-1007 (1965) . C . C . SCOTT, R . C . ANDERSON, and K . K . CHEN, J . Pharmacol . Exptl . Therap . _86 113-119 (1946) . E . E . FOLTZ, A . C . IVY, and C . J . BARBORKA, J . Lab . Clin . Med . _28 603-606 (1943) . C . J . PFEIFFER and G . H . GASS, Canad . J . Biochem . Phyaiol . _40 1473-1476 (1962) . W . KUIiLMANN, H . G . FROMME, E . M . HEEGE, and W . 05TERTAG, Cancer Res . _28 2375-2383 (1968) . S . EPSTEIN and H . SHAFNER, Nature _219 385-387 (1968) . M . KURATSUNE and W . C . HUEPER, J . Nat . Cancer Inst . _20 37-51 (1958) . P . J . DONOVAN and J . A . DIPAOLO, Cancer Res . 34 2720-2727 (1974) . J . SIMON, S . YEN,'and P . COLE, J . Nat . CancerÎnat . _54 587-591 (1975) . R . W . MORGAN and M . G . JAW , Can . Med . Asaoc . J . _111 1067-1070 (1974) . I . D . J . BROS S and J . TIDINGS, Prev . Med . _2 445-451 (1973) . E . L . GREEN, Am . Zoll . _3 358-360 (1963) . E . L . GREEN, Handbook on Genetically Standardized Jax Mice , p .3 Bar Harbor Pub . Co ., Bar Harbor, Maine (1962) . N . MANTEL, Cancer Chemother . Rep . _50 163-170 (1966) . 0 . STAUBELT, C . P . SIEGERS, H . BREWING, and J . STEFFEN, Z . Ernaehrungswias 12 252-260 (1973) .
