PROJECT REPORT COMMITTEE ON FOOD RESEARCH RESEARCH AND DEVELOPMENT BRANCH MIL1TAPY PLANNING DIVISION OFFICE OF THE QUARTERNA8TER GENERAL QUARTERMASTER FOOD ANO CONTAINER INSTITUTE FOR THE ARMED FORCES CHICAGO ILLINOIS COOP ERATI NG INSTITUTION LOCAL ITT Emory W« Thurston Laboratories Los Angeles, California DIVISION OEP ARTM ENT Research OFFI Cl AL I N VESTI GATOR COLL ABORATORS B. H* Ershoff REPORT NO. FILE NO. 7 N-1105 CONTRACT NO. W11-009-qra-70168 for period Covering 1947 - October, 1947 INITIATION DATE July 1, 1946 Tl TL E: PROGRESS REPORT PHASE REPORT ANNUAL REPORT IT E RMI NATION REPORT wEffect of Stress Factors on Nutrition” SUMMARY The Effects of B Vitamins, Liver and Yeast on Atabrine Toxicity in the Rat Immature female rats were maintained for eight weeks on purified rations containing 500 mg of atabrine per kg of diet, and the effects of feeding were contrasted with that observed on similar rations with atabrine omitted# Four experimental rations were employed: (1) a basal ration containing the B complex factors in synthetic form only (2) the basal ration plus ad- ditional B vitamins (5) the basal ration plus yeast and (4) the basal ration plus desiccated whole liver. The effects of atabrine feeding differed significantly on the various diets employed. Administration of atabrine plus basal ration resulted in a mark- ed retardation of growth, alopeoia, inhibition of ovarian development, en- larged submaxillary glands, granulocytosis and myocardial damage as indi- cated by electrocardiographic tracings. These affects were largely coun- teracted by diets containing whole liver or yeast ana to a lessor extent by the administration of additional B vitamins. On atabrine-free rations no abnormalities were observed on any of the diets employed. Desiccated whole liver was more effective than yeast or the additional B vitamins in promoting growth and ovarian development in the immature ata- brine-fed rat. The protective faotor(s) was present in the water-insol- uble fraction. of liver remaining after removal of the extraotable water- soluble material. An increased B vitamin requirement was observed in immature rats fed toxic doses of atabrine. The suggestion ic made that in addition to the known B vitamins still other factors are present in whole liver and yeast that are required in increased amounts in the atabrine-fed rat* N-1105 7 1 CQHO FORM 5 April 46 12-121 (i«vimmd) RESTRICTED Introduction Available data indicate that in addition to the major nutrients, substances are present in our diet which may be required in increased amounts during con- ditions of stress# Such factors are apparently dispensable under normal con- ditions or their requirements are so small they may readily be met by amounts present in the diet or through the synthetic activity of the intestinal flora or the animals• own tissues# Certain drugs or other "stress factors" may, how- ever, increase requirements for these substances to the extent that deficiencies occur, manifest by retarded growth or tissue pathology, and preventable by the administration in appropriate amounts of the missing nutrient (Ershoff, *47s }# In the present communication data are present indicating that atabrine is such a stress factor# Administration of this drug in toxic amounts resulted in the young rat in retardation of growth, alopecia, inhibition of ovarian develop- ment, granulocytosis and other pathological effects preventable, at least in part, by the administration of an unknown nutrient present in liver and yeast# Procedure and Results Four basal rations were employed in the present experiment: diets A, B, C and D# Diets A and B were purified rations containing the B complex factors in synthetic form only. Diets C and D were similar in composition but contained yeast or desiccated whole liver in addition to the synthetic vitamins# Sixty*- four female rats of the Long-Evans straii were selected at 21 to 23 days of age and an average weight of 41.8 gm# for the present experiment# Animals were kept in metal cages with raised screen bottoms to prevent access to feces, and were fed ad libitum the diets listed in Table 1# Feeding was continued for 8 weeks or unFil death, which ever occurred sooner# During the eighth week electro- cardiograms were taken of all rats. Animals were autopsied on the 56th day of feeding; ovarian and submaxillary weights were determined; and sections of the ovaries, submaxillary glands, liver and heart prepared for microscopic study. Tissues were fixed in 10$ formol and stained with hematoxylin and eosin# (see ond of paper) Table I Findings indicate that the toxic effects of atabrine in the immature rat may be counteracted by dietary means# pathological effects of atabrine were most pronounced on the synthetic ration, diet A, and least evident on the liver-con- taining diet D# Atabrine-fed rats on diet A exhibited a marked retardation of growth (tabla |l), extensive alopecia, infantile ovaries and enlarged submax- illary gland# (table II), granulocytosis (table III) and electrocardiographic abnormalities# Three of the 10 rats on this diet failed to survive the experimen- tal period of 8 weeks; and of the remaining 7 animals only 2 gained weight after the second week of feeding# Eight rats in this series developed extensive alope- cia during the first month of feeding although new hair had generally replaced the areas of alopecia by the eighth week# At autopsy ovaries appeared infantile both in weight and microscopic appearance. The most striking finding, however, was a marked hypertrophy of the submaxillary glands# These weighed 3 to 4 times more than those observed in normal animals of simialr weight and were signifi- cantly heavier than those observed in the much larger atabrine-free controls (table II)# Histologically the increas 1 size of the submaxillary glands appeared to be due almost entirely to hyperplasia and hypertrophy of mucous cells.^ 1 We are indebted to professor E*M* Hail, Dopt* of pathology, Univorsity.of Southern California Medical School, for the examination of the histological ma* terial. N-110S #7 2 Retardation of growth was similarly observed in atabrine-fed rats on diets B, C and D although growth was significantly greater on these rations than on diet A, Atabrine-fed rats gained most weight on the liver-containing diet D with growth somewhat less on diots containing yeast (diet C) or the additional B vitamins (diet B#) Three rats on the latter ration plateaued in weight after the first few weeks of feeding; the remainder of the group, however, and all atabrine-fed rats on diets C and D gained weight consistently during the exper- imental period# With the exception of the 3 rats on diet B that plateaued in weight, alopecia was not observed in any of the atabrine-fed rats except those on diet A« Ovaries appeared infantile both in weight and microscopic appearance in all atabrine-fed rats on diet A# They were somewhat larger in atabrine-fod rats on diets B and C but were still signifioantly smaller than those observed in ata- brine-free controls, On diet D no significant difference in ovarian weight was observed between animals fed atabrine and those on similar rations with atabrine omitted; and histologically the ovaries of atabrine-fed rats in this group appear- ed normal in all respects. On rations free of atabrine ovarian weights did not differ significantly on any of the diets employed; and histologically ovaries ap- peared normal in all groups (table II,) Enlargement of* the submaxillary glands was directly correlated with alopecia and failure to gain weight# Enlarged submaxillaries were only observed in ata- brine-fed rats that lost fur and plateaued in weight (8 rats on diet A; 3 on diet B), with glands apparently normal both in size and histological appearance in re- maining animals of the atabrine series* - In no instance were submaxillary glands enlarged on atabrino-free rations (table II), Table II (see end of paper) Per cent and total granulocytes per cc. of blood were markedly increased in atabrine-fed rats on diet A# During the sixth week of feeding total and differ- ential white cell counts, hemoglobin determinations and total red cell counts were made on the tail blood of all surviving rats# Differential counts were made on smears stained with Wright*s stain, 100 cells on each of 2 slides being em- ployed for each analysis* All blood counts were made in duplicate. No significant difference in total erythrocytes or hemoglobin levels was observed on the various diets tested between animals fed atabrine and those on atabrine-free rations# Erythrocytes averaged 7#3 to 8#1 million per cc, of blood for the various groups (range 6#4 - 9#3 million per cc#), with hemoglobin averag- ing 15,4 to 16,1 mg,/100 cc# (range 14*6 - 17«2 mg./lOO co.)* Total leucocytes per cc# of blood did not differ significantly on the various rations tested; a significant increase in per cent and total granulocytes per cc# of blood was ob- served, however, in atabrine-fed rats on diet A# Such was not the case with atabrine-fod rats on other diets tested nor for animals on atabrine-free rations (table III), Table III (see end of paper) N-1105 #7 In agreement with earlier findings (Hegsted, McKibbin and Stare,’44) no consistent abnormalities were observed histologically in the liver and myocardium of atabrine-fed rats on the various diets employed, nor did these tissues differ significantly from those of animals fed similar rations with atabrine These findings are in contrast to those of Wright and Lillie (’43) and Siegel and Mushett (’44) who observed neorosis and replacement fibrosis in the liver and myocardium of atabrine-fed ra-'-s* These differences may be due, at least in part, to the amount of atabrine fed, the composition of the diets employed or a strain difference in response to atabrine feeding* Myocardial damage in atabrine-fed rats was indicated, however, by means of electrocardiographic tracings* Electrocardiograms were taken with a Cambridge- Hindle 2 galvanometer electrocardiograph (research typo unit) of all surviving rats during the eighth week of feeding* Resistance was standardised for each animal, and the standard three leads wore taken on unanesthotized rats at a paper spoed of 100 cm. per second2. A marked elevation of the ST segment (indicative of myocardial damage) was observed in the electrocardiographic tracings of 5 of the 7 atabrine-fed rats on diet A and 5 of the 9 atabrine-fed rats on diet Elevated ST segments did not occur in tracings obtained from rats fed diets C2 or Dg or from animals fed atabrine-free rations* Further abnormalities in the ata- brine series consisted of a prolonged PR interval (indicative of delayed AV con- duction) in animals fed diet Dg# Six of the 10 rats on the latter ration had a PR interval of 0*05 seconds or longer in contrast to an approximate value of 0.04 seconds in virtually all rats fed other rations tested* With the exception of the above, electrocardiograms of atabrine-fed rats did not differ signifi- cantly from controls either in comploxes or in ventricular No abnormal- ities were observed in the electrocardiograms of atabrine-fed rats on diet C* In subsequent work experiments were conducted in an attempt to concentrate the factor or factors in liver responsible for its protective effect* Immature female rats of the Long-Evans strain were weaned at 21 to 23 days of age and fed ad libitum the following 3 diets: (1) diet A (2) diet A plus Liver Concentrate Powder 1-20 (Wilson) added at a level of of the ration and (3) diet A plus Extracted Liver Residue (Wilson) added at a level of 10%* The liver fractions were added in place of an equr,l amount of All diets were supplement- ed with 500 mg* of atabrine p .• kg* of diet* Feeding was continued for 8 weeks (10 animals per group*) Findings indicate the protective factor or factors is either water-insoluble 2 Yfe wish to express our sincere appreciation to Dr. John C. Ruddock, for the examination and description of the electrocardiographic tracings* Dr. Rud- dock is Clinical professor of Medicine, University of Southern California Medical School, Chiof of Medical Service and Head of the Department of Car- diology, St# Vincent’s Hospital, Los Angeles, California. 3 ST segments were elevated on all 3 leads on diet Bg and on leads II and III on diet Ag. Slight elevations of the ST segment wore occasionally notod in leads II and III on all control rations and on diets Cg and Dg. In no case, however, were they so pronounced as those on diets Ag or Bg. 4 Hoart rates averaged 504 to 537 per minute for all groups with an individual range of 465 to 570 per minute. The Q-RS interval averaged 0.02 seconds for all groups. 5 We are indebted to Dr. David Klein of the Wilson Laboratories, Chicago, 111., for the Liver Concentrate Powder 1-20 and the Extracted Liver Residue employed in the present experiment. N-1105 #7 .>r is chemically bound so that it may not be readily removed by simple water jxtraction* No significant difference in growth or gross appearance was ob- jerved between atabrine-fed rats on diet A and those receiving Liver Concentrate powder 1-20 (containing the water-extractable material of raw liver.) On the >ther hand, Extracted Liver Residue (consisting of the coagulated, water-insol- tble material remaining after the removal of the extractable water-soluble lubstances) was virtually as effective as whole liver powder in counteracting ftabrine toxicity in the rat* Animals fed the above rations gained the follow- ing amounts of weight during the 8 woek feeding period; diet A, 41*6 10*4 .pis; diet A plus Liver Concentrate Powder, 52.8 i 9*7 gms; diet A plus ex- tracted Liver Residue, 92*74y, 9*3 ms. Discussion Available data indicate that factors are present in liver and yeast that will counteract, at least in part, the effects of drug toxicity in the rat* As early as 1922 Funk («22) expressed the view that the composition of the diet and perhaps its vitamin content may have a profound influence on the toxicity of drugs* This suggestion has been amply confirmed, not only in regard to the known nutrients (Ershoff, *47a) but to additional factors present in liver or yeast. The beneficial effects of the latter in animals inhaling carbon tetra- chloride or fed toxic doses of strychnine, promin, dinitrophenol, sulfanilamide and other drugs has been recognized for years (De Santirrnez, *47; Battelli,»40; Higgins,»44; Chamelin and Funk, *43)* Similar results have been observed follow- ing toxic doses of diethyl-stilbestrol (Funk and Funk,*39; Chamelin and Funk,*43), estrogen (Engel and Rosenberg,*45; Ershoff,*47b,c)• Tho present experiment in- dicates that atabrine toxicity may also be counteracted, at least in part, by the administration of whole liver or yeast* Findings indicate that the offocts of atabrine administration in the immature female rat are dependent on the diet employed* With rats fed a synthetic ration (diet A), administration of atabrine at a level of 500 mg per kg of diet resulted in marked retardation of growth, alopecia, inhibition of ovarian development, enlarged submaxillary glands, granulocytosis and myocardial damage as indicated by electrocardiographic tracings* The above effects were largely counteracted the administration of desiocated whole liver at a level of 10% of the diet in placo of an equal amount of sucrose* On this latter ration (diet Dg) growth was significantly greater than on diet Aoi alopecia did not occur; ovaries and sub- maxillary glands appoared normal both in weight and microscopic appearance; granulocyte counts wore normal; and electrocardiograms wore free of the abnor- malities observed on diet Ag although some prolongation of the FR interval was observed* Similar results wore obtained with diet Cg (in which yeast was fed in place of the whole liver) although growth was loss than on diet Dg end ovaries resembled in weight and microscopic appearance those of an immature rat* Elec- trocardiograms in this series, however, remained free of the abnormalities ob- served on other atabrine-containing rations and were indistinguishable from those of normal controls. Findings on diet B2 were intermediate between those on diet Ag and rations containing whole liver and yeast* Threo of tho 10 rats on this diet failed to grow; they developed alopecia and at autopsy revealed markedly enlarged submaxillary glands* Tho romainder of the series, however, were indis- tinguishable grossly or in microscopic appearance from animals fed yeast (diet Cg)* Electrocardiographic tracings revealed a marked elevation of the ST seg- ment similar to that observed on diet Ag in 5 out of the 9 rats on this ration* On atabrine-free diets no abnormalities were observed on any of the rations em- ployed. N-1105 #7 Since the toxic offects of atabrine wore less pronounced on diet B£ than on diet Ag and since these two diets differed only in their content of known B vitamins, it is apparont that tho beneficial offects of diet Bg wore due to its increased contont of B vitamins* The latter ration contained twice the thiamine, riboflavin, pyridoxine, pantothenate and niacin content of diet A as well as significant amounts of biotin, folic acid, inositol and p-aminobonzoic acid* It would appear that one or more of the latter factors were responsible for the ob- served effects# The toxic effects of atabrine wore still, however, more pro- nounced on diet Bg than on yeast or whole liver-containing rations (diet C2 or Dg)• Those findings would indicate that in addition to the known B vitamins there were additional factors in the whole liver and yeast that counteracted, , at least in part, tho effects of atabrine toxicity in the rat# preliminary re- sults indicate the protective factor(s) is prosent in the water-insoluble fraction of liver remaining after tho removal of the extractable water-soluble material* References Ba-btolli, G. 1940 Detoxicating action of livor extracts in experimental strychnine poisoning# Boll* soc. ital. biol* sper., vol* 15, p* 687; Chom* Abstr. 1946 vol. 40, 58339 Chamelin, I*M. and C. Funk 1943 The action of liver extracts in counteracting the toxic effects of diothyl-stilbestrol and sulfanilamide. Arch* Biochem*, vol. 2, p. 9 Do Santibanez, J.D* 1943 The detoxifying principle in livor, Rev. facultad. cienc. quim. vol. 18, p. 177; Chem Abstr. 1947, vol. 41, 10401 Engel, p. and E. Rosenberg 1945 Estrogen-inactivation liver extracts. Endocrin- ology vol. 37, p. 44 Ershoff, B.H. 1947a Conditioning factors in nutritional disease, physiol. Rev., in press Ershoff, B*H. 1947b Effects of liver feeding on growth and ovarian development in the hyperthyroid rat. proc. Soc. Exp. Biol, and Med., vol. 64, p. 500 Ershoff, B.H* 1947c Comparative effects of liver and yeast on growth and length of survival of tho immature thyroid-fed rat. Arch. Biochem., in press Ershoff, B.H* and H.J. Deuel, Jr* 1946 The beneficial effects of yeast on the body and gonadal weight of immature rats fed alpha-estradiol. Amer.J.physiol•, vol. 145, p. 465 Ershoff, B.H. and D.Horshberg 1945 The beneficial effects of yeast on the cardiac failure of hyporthyroid rats. Amer. J. phyiol., vol. 145 p. 16 Funk, C* 1922 Die Vitamie, II Ed., Bergmann, Wiebaden, p. 341 Funk, C. and I.C. Funk 1939 The action of certain hormones as dietary .con- stituents. Science, vol. 90, p. 443 Hegsted, D.M., J.M* McKibbin, and F.J. Stare 1944 Nutrition and tolerance to atabrine. J. Nutrition, vol. 27, p. 141 Higgins, G.M. 1944 The influence of purified diets u .,:i the toxicity of promin. Am. J. Clin, path., vol. 14, p. 278 N-1105 #7 6 Siegel, H# and C.W* Mushett 1944 Structural Changes fo_lowing administration of quinacrine hydrochloride* Arch* path*, vol* 38, p* 63 Sure, B. 1941 Dietary requirements for fertility and lactation. XXIX* The existence of a., new diotary factor essential for lactation. J. Nutrition, vol# 22, p« 499 Wright, C#I# and R#D* Lillie 1943 Toxic effects of atabrine and sulfadiasine in the growing rat* public Health Rep., vol. 58, p* 1242 N-1105 #V Table 1 Composition of Experimental Diets Dietary component Dlot A1 1111,1 A2 Diet B. and B„ 1 2 Diet and Cg Diet and Dg Yeast* 0.0 0.0 10;0 0.0 Whole Liver Powder^ 0.0 ' 0.0 0.0 10.0 Vitamin Test Casein** 22.0 22.0 22.0 22.0 Salt Mixture4 4.5 4.5 4.5 4.5 Sucrose 73.5 73.5 63.5 63.5 Atabrine (quinacrine hydrochloride)® was incorporated in diets Ag,B2# C2 and Dg at a levol of 500 mg* per kg* of diet, replacing an equal amount of sucrose. To each kg* of diet A, C and D were added the following synthetic vitamins* thiamine hydrochloride 72 mg*, riboflavin 9 mg,, pyridoxine hydrochloride 15 mg*, calcium pantothenate 67*2 mg., nicotinic acid 60 mg*, 2-mothyl~naphf* thoquinone 5 mg*, and choline chloride 1*2 gm* To each kg* of diet B were added thiamine hydrochloride 144 mg,, riboflavin 18 mg*, pyridoxine hydrochloride 30 mg*, calcium pantothenate 134*4 mg,, nicotinic acid 120 mg*, inositol 1*2 gm*, p-aminobenzoic acid 600 mg,, folic acid 10 mg*, biotin 1 mg*, 2-me thy 10 mg* and oholino chloride 1*2 gm* Each rat also received throe times weekly the following supplement; cottons seed oil (Wesson) 500 mg*, alpha»*tocophorol 1 mg,, and a vitamin A-D concen- trate® containing 50 U*S«P* units of vitamin A and 5 U,S*P* units of vitamin D* —, I—- - , I , , ,—,—" ■—» '—' Footnotes to Table 1, 1 Brewer*s Type Yeast No* 200, Anheuser-Busoh, Inc,, St, Louis, Mo, 2 Whole Dried Liver powder. Armour and Co,, Chicago, 111, 3 Vitamin Test Casein, General Biochemicals, Inc,, Chagrin Falls, Ohio 4 Salt Mixture No* 1 (Sure, *41), * 5 Quinacrino HCl powder (Atabrine), Winthrop Chemical Co*, New York, N,Y, 6 Nopco Fish Oil Concentrate, assaying 800,000 U#S»P, units of vitamin A and 80,000 U*S*P« units of vitamin D per gram# N-1105 #7 8 Table II Effects of atabrine on growth and ovarian and submaxillary weight in the immature female rat $iotai*y Numbeh group of animals Initial body weight Gain in body wt. over 8 wki period Average ovarian wt Average sUbmaxil- lary wti^- Ratio of sub-* maxillary wtt to body Wt4 10-3 i Atabrine Series V. ” * gm. gnu mg. 1 A 10 41*7 33.9 ± 12.1 - 12.4 > 3.4 486.4 4 41.6 6.43 * (V B 10 41.9 87.4 4- 10.8 26.0 43.5 418.6 4 39.4 3.24 r. : (9) , Q 10 41.8 99.1 4- 5.1 30.4 ± 2.7 303.8 4 10 *8 2.15 (10) • D 10 41.8 118.2 ± 5.2 43.5 t 2.9 317.64 19.4 1.92 (10) < Atabrine- free Controls A 6 41.7 146.4 +- 9.0 47.0 4- 2.4 374.8 4 18.4 1.98 (6) B 6 42.0 152.34 7.8 49.1 4 3.0 334.6-fc 16,8 1.72 (6) C 6 41.5 146.84- 6.9 43.2 4 3.2 317.3 4 32.8 1.69 * (6) , D 6 41.7 169.7 4 9,3 44.8 4 2.1 313.5 4 14.8 1.49 (6) The values in parentheses indicate the number of animals which survived and on which averages are based. Footnotes to Table II* 1 Including standard error of the mean calculated as follows: /£d2 / /rr / J n where MdM is the deviation from the mean and wnM is the V number of observations. N-1105 #7 Table III Effects of atabrine on the granulocyte count of the rat. Dietary Number j group of animals Total leucocyte count Average*- Range Granulocytes per cent* Total*- per cc. Atabrina Series A 9 17,090 1,540 10,200 - 24,800 43.9 iz 4.3 7503 -b 735 B 10 15,900 1,310 8,600 - 24,100 23.0 ± 1.7 3657 ± 270 C 10 13,280 1,650 9,200 - 20,400 24.3 4- 2.5 3227 ± 332 D 10 12,270 1,280 7,900 - 18,400 21.5 ±2.3 2638 ± 282 Atabrine-free controls A 6 13,150 860 8,800 - 15,800 18.6 4*.2.6 2446 ± 342 B 6 12,870 1,040 7,800 - 18,100 17.4± 2.8 2239 t 360 C 6 14,180 810 8,200 - 16,600 19.1 ±1.6 2708 ± 227 Footnotes to Table III. 1 Including standard error of the mean calculated as follows: / L d2 / , I f n where "d” is the deviation from the mean and unw is the J number of observations. N-1105 #7 10