fHE w s o f cr m i x ( w m m s s Meh of these metal %m&$ if any, is concerned with the “natural ensyma1* can net be said at this time* vii TABLE W GCNTIBTS Page xmoimifOT*. ***....***.**.* *..****.....*....... **..****. IttSTGRIGAL* .A***.**........,*.*****... *..... *....*....*.... i 3 Occurrence of Cadmium*.♦***..••..*.*.... 3 Cadmium Poisoning in Han***.*.**,.•*••«•*•••.... •«•*••*••*••• 1* lisperlmental O&dmlm Poisoning*••*•**•«*.*••••*««.*•••....*** 6 Effect on B n a y m e g * . * ♦. lli Cadiaium in Drinking Water****.•»*•••*•*•••••*•**•»*•««*. 18 ..... * 22 EXPE&IHEOTAL ...... Part 1* Chronic TcadLelty Experiments* Eat Care and Diets..*.....*...*............*...*..**.*..... Blood Studies**•*.*••*•*..**•*♦**.*..*•.*..***•*.... Pathology.** ************* •**•««••** Cadmium Analyses..*.*.***.**.*#..... Part IX* lasym© Studies*. ... Preparation of Pormyl^L^lutamie Acid*•*«**••*•••***••«•*•• Isolation of Fer®yl^^lwfla»oic Acid Deforirylase*........... Determination of Easymabie Activity** Determination of Glutamic Aeld* ... *..... ..... ******.... Formic Acid Betermliiation* Protein Determination. Electrophoretic Studies. ******* RESULTS AND CCKI^SICKS**,****.*********.***..... Chronic Toxicity Study Cadmium Analyses of Liver andKidney Enayme Studies*****.********.*.* SM1AEX.*.* BXBLIOGBAPHI...... ......... .... .... 22 22 2$ 25 2$ 28 28 30 32 33 3h 3k 36 37 37 hk ***** $0 Si* *...... $6 LIST CF TABLES TABLE X Conposiilon of Diet A Page . , . . , , . . . . X X XX Initial Average Group I t e i g h t a . . . . * . . * . . . . . 22 23 XXX torposition of the Hcp|>erfc~Hmi Diet***.*.***.****...... ♦•**•****.**#**. 2k IV Cadmium Content of V Growth of Rate Receiving g0 ppm Gadto&nm— Groi^j VII. .......* 2h VI Ghrosnatography of L~GXutamic Acid and Porinyl-L-Gluiainlc Acid .... VXX Average Body Weight, of Each. Group*. VIII Average Weight Gain of Each G r * » # o , u ♦ 29 3 p 8 39 , XX Average Food C o n e m i p t i o n . * . , . 1*0 X Average Water Intake of Bach Groijp,.. . . . . . . JUl XX Average Cadmiim Intake of Each Croup....................... 1*2 XXX Gadmim Concentration in Bat Kidney,.......**...#*.......*. h$ XXIX Cadmim Concentration in Bat Liver......................... 1*6 XXV CadtedLm Retention After Gm Year........................... 1*7 XV Purification of Fcntyl-L^OIufcaffiic DeforayXase 51 XVI B&symtle Activity..*.* * « # . * * # * # # ♦ * * * * . $ 1 1 immfocfxm Cadmium has recently been found to occur in small concentrations in some ground water*! however little experimental evidence is avail­ able concerning the effects of prolonged Ingestion, by animals or smm, of water containing trace quantities of this metal* In cases of industrial cadmium poisoning, both acute and chronic, and from experiments carried oat with animals, it has been found that cadmium eratur© maintained between 75 to 78°C. All animals received ad libitum the Hoppert-Hnnt stock diet shown in Table III* TABLE III COMPOSITION OF THE H««T-HtmT DIET Constituent Ground yellow corn meal Ground whole wheat Powdered whole milk Linseed oil Alfalfa Brewers yeast Sodium chloride i Per Cent by Weight 32.5 25.0 22.5 10.0 6.0 3-0 1.0 The control group (Group I) was given distilled water ad libitum* The other groups were given water containing different quantities of cadmium ad libitum, as shown in Table IV, prepared by diluting a stock solution of cadmium ehlorAd© with distilled water* Vfeekly records were kept of body weight and food and water consumption* At the end of the first slat months period one male and one female from each group, I through VI, were sacrificed* Tissues were taken for pathological studies and for cadmium analyses* The rest of the animals in each group were sacrificed at the end of a year*s time* Animal© which died daring the experimental period were examined for gross pathological changes, and, in some cases, tissue sections were prepared* 2h TABLE 1 7 caihiim G o m m u m m t m Group Humber Cadmium Concentration I 11 1X1 tv 7 71 0,0 0*1 Q*5 O Cf 5.0 10.0 Cadmium chloride in distilled water calculated as parts per is&llion (ppm) cadmium ion* A grcfup of rats (Group 711)cassisting of eight males and eight females w#®. given water containing $0 parts per million (ppm) cadmium^ all other treatment of these animals was the same as above, with the exception that the. rats were sacrificed at the end of three months. Initial and final body weights of these animals are given In Table 7* TABLE 7 gbgwth m m m m m x m m so im tu ^erageteo^-_j^h|t[ Initial V^ight Kales Females £g*i___ ___ (g»?... ... ^ 73 89 137 11*7 81** Males Females Males females (e-1 _ _ -... (g.> 211 193 21*6 215 Average Vfeight Gain g,/rat/day 3*1 2*7 Bajt6 after essperiaent began. 2*5 1*6 1.0 0.6 25 Blood studies Included determinablone of the total red and white ©ell ccnmts* differential white ©ell ©omit and the determination of the hemoglobin coneentration by a modified Sanford method (?8). Blood analyses m m made at monthly intervale m four males and foar females in each eaqperianatal grcep and on five males and five females in the control gr©\$>* Bats at the age of six months and one year were killed with one<3uarter ml. of H&Xatal (sodlum-ethyl (l-msthyl-butyl) barbiturate) in the thoracic cavity* Samples of the following tissues were taken* kidneys* adrenal glands* liver* spleen* heart* brain* stomach* duodenum* Ileum* colon* and cross-section of bone marrow of sternum and femur. Fixatives were 10$ saline-formalin and Camay1s fluid for glycogen* All of the tissues were stained with hematoxylin and eosin. Small portions of the liver* kidney and adrenal gland were stained with Best*© Carmine for glycogen and Sudan 1? for fat* Weights of the liver* spleen mid kidneys were recorded. liver* kidney and bone sassples were frozen m solid carbon dioxide and kept in the deep freeae at -X5°C until analyses were performed. Whole liver and kidney sables were wet ashed in Phillips beakers* ^un All pathological studies were performed by the Department of Animal Pathology* M. S. II,, under the direction of Dr. Robert F, Langham. 26 It m s found most satisfactory to add fifteen ml. concentrated sulfuric acid and fifteen ml, concentrated nitric acid to the tissue, then cover the beaker, and allow it t© stand overnight. The next day the beaker was heated on a hot plate; ten ml. portions of nitric acid were added until no more charring occurred, and the sample was almost colorless* The volume was reduced to about ten ml, by strong heating and after eOH&ilng the beaker, the samples were titrated with sodium hydroxide to a yellow color using thymol blue indicator (pH 2*8)* (This step is critical to obtaining low blanks and accurate results*) The sasple was then diluted to 25 ml. with distilled water, and, depending on the expected cadmium concentration, either whole samples were analyzed or aliquots were taken* Saltzman* s micromethod for cadmium (79) was used* This method has the advantages that it can be applied to sasples contain­ ing as much as 5 to 10 mg* of common interferring metals and that no additional purification of reagents is necessary* The procedure consists of extracting the cadmium with diphenylthiocarb&zone (dlthifcme) in chloroform, removing the cadmium ion from the eadmium-dithisime complex by eosplexing the cadmium with tartaric acid, and then by making the solution basic, re-extracting the cadmium with dithizone* The optical density of the pink solution of cadmium dithi- zonate in chloroform was determined at 515 mp, in the Beckman Model B, equipped with a special cell holder to accommodate Coleman matched tubes 15 mnu in diameter* A standard curve was obtained by carrying known concentrations of cadmium nitrate solutions through the whole 27 procedure # Recoveries were run on tissues with known coneentratione of cadmium nitrate added before the ashing procedure# Recoveries averaged 95 bo 98 per cent* Blank® were also run on the acids used in ashing the tissues# Lew values obtained for control tissue blanks were subtracted f r m concentrations of eadmim found in experimental tissues, the reagents used were 0* and were not further purified. Ordinary distilled water was used and no interferences £?m contaminating lone were noted* Uhdar our conditions the range of the working curve was f r m 0*2 microgrsm to 10 micrograms of cadmium in the final solution of 15 ml* chloroform. Over this range Be©rfs law was followed; in fact there was only a small deviation for a 20 microgram standard cadmium solution.* the sensitivity of the method was 0*05 micrograms in a volume of 15 ml* Contrary to the findings of the published method, it was noted that the final chloroform solution lost color upon, stand­ ing as llbtl© as three hours at room temperature. Readings were there­ fore made iasaedlately after each sample was carried through the extraction procedure. Rone samples may be analysed with the following modifications. Ten ml. of concentrated sulfuric acid end tea ml* of concentrated nitric acid were added to a bone weighing approximately one gram. This mixture was heated on the hot plate, no longer than an hour and a half. At this time the solution was colorless, but there was a large amount of pre­ cipitate in the flask. After cooling the flask, 10 ml. of water were added and the sample was titrated with concentrated ammonium hydroxide to a yellow color with phenol red (pH 8*3). SoUd sodium citrate was 28 thee added with constant swirling, mtil all the precipitate was in solution, (this usually took shout 16 grams of sodium citrate 239^0) for a one gram heme#) The clear solution was trans­ ferred to a separatory funnel, and 10 ml. of dithizone in chloroform (the same concentration as was used in Saltaman*s procedure) was added. After shaking the funnel, the chloroform layer was drawn into another separatory funnel containing 30 ml. of 1 H HOI* the solution in the first separatory funnel was rinsed with 10 ml* of chloroform and after shaking the funnel, the chloroform layer was again drawn into the second funnel. After shaking the second funnel, the chloroform layer was discarded, and the solution was washed with 10 ml. of chloroform, which was also discarded. The solution of cadmium chloride was then neutralised with $JaOH to the thymol blue endpoint (pH 2*8} as described previously, and the solution carried through the usual Saltsmsn pro­ cedure. Part IX* Basyme Studies Preparation of Formyl-X-glutamic Acid Fcrmyl-l-glut©mio/waldprepared from formic acid and L-glutamie acid according to Tabor and Mehler*s procedure (6?). It was found that the ethanol-benzene solution of forayi-L-glutamic acid described in the procedure must be left in the cold room at 5°&* several days in order for crystals to form, and that the beaker must be scratched occasionally during this time* Difficulty was experienced in trying to remove the benzene completely from the acid. The benzene was finally removed by 29 lycphilizaticn of the confound for several hours. The melting point was 112°C* j (m, pt# found by Tabor was 112°C.) and when mixed with 1 known fcr^l-L-glutastlc acid there m s no depression of the melting point-* Ascending chromatograms were ran in the following solvents and the following values were found, which agree well with mines given in the literature (67*80). tpattTt? TTY emcmTOGEAEHr m l-csujtakic acid ahd fgrmsx-l-qldtamic acid Oostpoiiud t-Butanois’ Hf ¥alue Solvent formicacid* fiScf' tiaenoil: &oO 70115*15 75*25 Fomyl-L-glutamic acid OmJk o*6i L-glutamic acid 0.14* 0*31 Detection of L-gluhamic acid and foa^yl-l-glutaMc acid cn the paper was accomplished. as fellows# After air drying the chromatogram to remove the solvent, the paper was placed in a Jar with a small amount of con­ centrated HC1* After two hours the paper m s removed and allowed to remain overnight la air to remove the HG1 fumes* Both glutamic aeid and formyl glutamic (hydrolysed to glutamic acid by the HCl) were visualised by spraying with ninfcydrtn. T"JI — ;...1.... . Obtained through the kindness of Dp* Herbert Tabor, national Institutes of Health, Bethesda, Hd. 30 Isolation of Formyl~L^lutamic Acid Befon^yXase 1 Albino guinea pigs weighing from i^OO to 700 grams were injected intramuscularly with a suspension of L«*hlstidine monohydrochloride2 in Masola (earn) oil* Che hundred rag. of histidine were injected per 100 grams of body weight* The animals were sacrificed 18 hours later by a blow on the head* The liver m s removed and an acetone powder prepared immediately by homogenizing the fresh liver for m& to two minutes in a faring Blender with five volumes of sold acetone (cooled to -30° to 4iO°C* with solid carbon dioxide)* The temperature of the preparation o stayed batman 2 to 5 0* although the entire operation was done at room temperature * The suspension was rapidly filtered with suction using a Buchner funnel and Whatman no# 1 filter paper, and the precipitate was washed with two volumes of cold acetone, The solid was not allowed to dry on the funnel, but- was rehmogeniaed with five more volumes of cold acetone, filtered and washed m. the filter with two volumes of odd acetone* The filter cake m s allowed to stand on the funnel until it m s nearly dry and was then removed to a large sheet of Whatman no* 1 filter paper and air dried after crumbling with the hands* The powder was then kept in the deep freeze at **2G°C* until needed. The yield of acetone powder was 31 .6$ (18 grams of acetone powder from 57 grams wet might of liver). ^The" guinea pigs were obtained through the kindness of Ik*. L. P. Hedeman of the Michigan State Health Department, Lansing, Michigan. \ (*) histidlne-monohydrochloride, C* P. (monohydrate) * Pfanstiehl Chemical Company, Waukegan, Illinois* 31 An initial attest to isolate f©rsyl-L~gXuiemle deformylas© by tbs procedure of Suda et aL* (60) resulted in a preparation with no activity- Therefore, various modifications of the preparation were tried* The procedure outlined below resulted In an acetone powder of the highest activity* All solutions were made up in glass redistilled JZ water* The acetone powder was extracted with SO volumes of £ x 10 M phosphate buffer, pH 5*6. The powder and buffer were rubbed together in a mortar and pestle* Tbs mixture was transferred to a beaker and stirred slowly with a mechanical stirrer for1two hours at room temperature* This mixture was then centrifuged* A two per cent sola1 tion of protamine sulfate was added with continuous stirring until the rati© of the optical density of the supernatant at 280 to 260 ran was about 0*8* The volume of protamine sulfate required was about 0*1 the volume of the crude protein solution. After centrifuging, the supernatant was heated in a beaker for ten minutes at 50°C. The selu­ tion was brought to 50 ° by rapidly heating the beaker in a water bath, with ccmtinuous stirring, during the ten minutes time* The mixture was then cooled quickly to 20°G* (la an Ice-salt bath) and centrifuged. The above operations except where stated otherwise were carried cut at room temperature* The following acetone fractionations of the protein solution were o carried out in the cold room at % C* One-half volume of cold acetone rrotamim sulfate, Nutritional Biochemicals, Inc* Two grams of pro­ tamine sulfate were dissolved in a small amount of water with the addition of two drops of 10 N NaOH, the solution neutralised t© pH g.O with acetic acid and made up to 100 ml. 32 {4i©°G*) asm added to the supernatant (the heat-treated enzyme) . A precipitate formed which m e centrifuged and discarded, A volume ut odd acetone time then added to the supernatant, equal to the volume of the supernatant and the mixture m s again centrifuged. The super­ natant was discarded, and the precipitate obtained from this second acetone precipitatlm was dissolved in a volume of redistilled water equal to that of the original extract. The mixture was centrifuged and the precipitate discarded. To me volume of the supernatant con­ taining the aeetone-fractimated enzyme, one-half volume of saturated arsftonium sulfate was addedj the pH was adjusted to 5*0 with 2$ per cent acetic acid.^ The beaker containing the solution was covered and left in the cold room overnight. The next day the mixture was centrifuged and the supernatant discarded. The precipitate was put Into water solution, in 0,1 the volume of the original crude extract, and the pH 2 adjusted to 7*0 with 1 H HaGK, The solution was dialyzed against redistilled water in the cold room for two hours. During this time the water was changed • CVI ra too 0> 1A V\ r < Os IN * -flt -sr «t m CO CM r f fri os> r-i 02 ca «0 CM § a eFv CO tW \© 5m CM «sr § •D CO r*M ® s*. sp f «* CM CM q CM |*M* CM ■8 I # rH 4s 1*5# I«2|f « N5T w **+*!? i ljffS# l •*‘•5# CM rH * ■+? °I ft 02 pH 8 St* <2. sa 8 CM ss Cm. CO w St CM US @9 &8 » CO V \ CM Os n tf CM (N* ISO CM R C *\ CA 8 pH CM CM fH CM CA CM O CM CM CM CM OK » CM OS rH CM H in R cr\ * „Cj is'S *£> pH (A 1 | 8 g © ** ° P* mJ OS H % A a f t **> IQ Q> **? i $ * m w d n tfp\ s© CM JS sit ?3 ^w 8 ® C ** r*H pH & rH tA PA CM «? ho w NO* a 1 3 St H * CM rH tA * CM rH NO pH H --S? pMH rH ■CO rH «*> 1A rH *r\ o rcHl a r-j rH 3 CO IN«H CO 4 rt rH * * NO | VO* H * # * rH a CO \f\ # CO o* o 00 rH 8 mSf O t ■nJ CO * p** rH «n rH 3 CA CA • INrH CO CO o> rH Nd rH 5 o CO V\ O 3 3 * a CO * 63 ft XA* «** © as rH m CM -4 rH ^# r ***- CA * * CO CO H pH CO r-r NO C— H o a ;3 A rH C H H* CN P— * « oq P * " 1 ' C O H rt H 'A © H 21 o\ H CO * CM « ®2 rH O p«** # CO rH # -=* f— * V\ o cq o a a 0\ * CO rH % 1 ~rH qf f \ *1 \ rH * CO a 4 rH {5» a Hi CO rH -sf # xr> rH £>• # CO rH u 12 J! 0 \ CM & « ?s & $ *j 5^ S\ £R ?5 «R cm tr\ r* H\ t wm m wmt s < ed> * mm aoraAV *TJ m *£ & ! £~ CJ s* PCM -* -CM ST e ** CM ♦ CO 00 CM C O C O OJ CM \D CM ffl Q) H p» £ «9 bZ TABLE XX AVBRAGB GAIMIUM BREAKS CT BACH GROUP Group Humber During Bxperimental Period pg*Cd*/rat/day* Total Intake for One Tear mg*Gd*/rai 0 0 XX 3*1 1*132 XXX 15*9 5.750 IV 82*1* 25.82 V 150*2 $1**36 VI 276*2 I 100*0 Cadmium (a© cadmium chloride) calculated from the water ccns*miptlc&* Bata for rats receiving 50 ppm of cadmium (Group VII) are presented separately in Table V* These rats were not pert of the chronic tcacicity experiments, tout were started in order to cospare the results obtained with a cadmium level which was thought t© toe high enough t© enable the rats to show gross signs ©f toxicity. It can be seen that, compared to the average weights of the first six grotps, rats receiving 50 ppm cadmium were stunted in growth* Teeth of seme of the rats receiving 50 ppm of cadmium showed the typical bleaching response as reported toy Ginn and Volker (32)* Rats frcaa Groups I through VI were checked periodically for this bleach­ ing, but no evidence of it was found* k3 Th© average water intake of the rats receiving $0 ppm of cadmium was mly ll^ ml* per day. this was roughly one-half of the water consuwption of the ether groups* The food intake of this group of rats was also less than that of the animals drinking lower concentrations of cadmium* The blood hemoglobin of rats in Group VII dropped within two weeks to 8*0 grains per 100 ml* of blood and stayed between 7*7 to 9*0 grams for the remainder of the three months period* Microscopic studies showed marked ©nisocytosis, with many* microcytic, hypochromic red blood cells and polychram&sia with eight to ten nucleated red blood cells per 100 white blood cells* The pathological changes described pertain to those of Groups I through VI. Pneumonia was the most important change observed xri all the various groups of rats* The disease was occasionally accompanied by a pleuritis and an empyema* The pneumonia appeared in the controls and various levels of eackaium as follows* Levels of Cadmium taiaber of Bats Number with Pneumonia For Cent with Pneumonia Controls 22 13 59 0*1 0.2$ 0*5 5*0 10*0 26 16 .16 16 16 18 12 12 11 12 69 Ih Ik 69 7k ppm ppm ppm ppm ppm In addition to the pneumonias* m e rat had a pleural tumor which was diagnosed as a squamous cell carcinoma and another rat had an undetermined nervous disturbance. The various levels of cadmium did hit not produce any recognizable microscopic changes in the various organs. Systematic studies of pathology were not performed on tissues from rats receiving $0 ppm cadmium, hut several animals were observed for gross pathological changes and none were noted# Cadmium Analyses of Liver and Kidney The tissue cadmium content of the kidney is given in Table XIX and that for liver in Table XIH# Analytical values fear tissue c&dteium la animals receiving cadmium for six months are given in terms of jag* cadmium per gram wet weight tissue, since portions of the live? and kidney were analyzed and weights of the whole tissues were not recorded# It will be remembered that the six months values were obtained from only two rats# therefore, averages of these groins do nest have as much significance as the values from the twelve months period. The values for tissue cadmium following twelve months exposure to cadmium are presented both as jug. cadmium per gram tissue and jag. cadmium in the whole organ* It can be seen that as the cadmium content of the drinking water increased, the amount of cadmium retained in the kidney incre&ed. In most cases, tissue cadmium concentration was roughly proportional to the increase in the cadmium content of the water* For example, ingestion of $»0 ppm resulted in a deposit of 52 jig. of cadmium in the kidney per gram tissue whereas 2#5 ppm resulted in an average cadmium content in kidney of 26 jag. The cadmium concen­ tration per gram wet weight of kidney at the end of a year was about 2*5 times as much as at the end of six months, whereas in liver the hS TABLE XXI 0AX6OTM COSGOTMTXClf IH BAT K UMM Group Humber I ppm Cd* in Mater Jtg*Cd*/g. Met Tissue Mb* 6 mmths 12 months* 0 0 0 u o a 0 1,68 (1,39-2.10) TTT imAmtt 0*5 k*$Q (1.72-7.35) 5.83 (2.1*U-8.91) 10.1 (7.25-12.9) {liuS>"40*2} ffi y ¥1 2*5 5*0 10*0 j&g.Cd* Total Organ 12 months 0 3 .M* 11.3 Z$*9 1*7.6 17.6 51*8 £ h *6 (12.8-22.3) (k9*k~$6*9) 30.2 (28.6-31.9 63*6 l?lt*0 (57.1-112*8) Values in parentheses are the range of values stained* # These are the average values f r m li, 5 or 6 rats in each grew# cadmium concentration at the end of the 12 months was about double the level at the end of six months* If one compares the jag* cadmium per gram in the kidney and liver, it will be seen that there m s more cadmium retained in the kidney* In fast, there was from two to three times the amount of cadmium re­ tained by the kidney as by the liver* Usually, however, the total cadmium content of the liver was equal to or greater than the cadmium level of the kidney, because of the larger weight of the liver. 1*6 TABLE XHI CUUHRM GOSCEHTRATIOH BJ BAT LIVER Qrmp jxg*Cd*/g* Wet Tissue Wfc« 6 months It months* Kttniber p|M in W&ter I 0 0 0*1 0 11 m , m V n jignCd# total Organ 13 months 0 0 0.23 (0.16-0.2?) 2.32 9.18 0 *5 0.50 (0.16-1.56) 1.09 (0.3U-1.22) t*$ 3.80 (3.67-3.91) 6.11 (3.71-10.3) 1*9.1* 5.0 7.50 (7.27-7.72) ' 16.3 (11.3-21.0) 11*1.0 lo.o 20.2 (13.6-26.8) 39.1 (27.8-58.8) 3U1*.8 Values In parentheses are range of values obtained. * These ere the average values from li, 5» or 6 rats la each group. Table 117 caspares the total concentration of cadmium present In the liver and kldaty and the per wart of the total ingested cadmium that each tissue retained. The per cent of the cadmium Ingested which was retained by both the kidney and the liver ranged from 0.3 to 0.5 par cent. The percentage of eaaulum retention was also calculated from tissue analyses of the rats which received 50 ppm. These values are nob directly censurable with the other groups, since tee rate receiv­ ing 50 ppm accumulated the element for only three months, whereas the values for the other groups were obtained at the end of a year's 1*7 & 4 rj rl l/\ fr" £ Xf\ On * CM Q2\ S n o* ov 4 j « &n Bn Bri 3 ri o* oj* o♦ o P'S VO O «M £-< *- O E-t *(■£ } JU Q m Cj fi4 «rH ..n .. * ■3 -at 43 C S C> M * m* o o * O o -Os at -al (*»» r-t Q U\ o* Os CM S# ^* 5♦ o o o o €h -at ***S c rq i * * e\j C\ 3 cm M d H & O * 73 CO 3n us * sp ST CD fcs* 4s ii8 exposure# However, the data do show that, except after administering 0*1 ppm. ©f cadmium, the kidney retained about the same Sper cent of m m m m Mrmh m * the Ingested dose regardless of the ©oneentration of cadmium in the water* C& the other hand, the per cent of the ingested cadmium stored in the Hirer seems to Increase with an Increase ±n dietary cadmium* Fooled samples of blood from rats receiving $0 ppm were analysed f m cadmium content# The concentration found was 11*5 Mg# per 100 ml of blood* If one assumes a blood volume of 6*? ml* per 100 grams of body weight (86), then these rats, when sacrificed, would have had a blood volume of ll* to Id ml*, and the cadmium content in their total blood volume would be only l*h to 1.6 jug* per rat* Because of the difficulty of obtaining enough blood from the rats on the chronic toxicity study, no blood cadmium analyses were made on these groups, but It can be assumed from the results given above that the cadmium concentration of the blood would be insignificant, except perhaps la rats receiving the 10 ppm of cadmium in water. trm the results of the chronic toxicity experiment, it can be seen that the presence of cadmium in the drinking water had no effect cm growth rate, food consumption, water ccasaaption or blood composition* This finding was net unexpected since the levels of cadmium were deliberately chosen to be those which might be as high as would occur in drinking water, but not high enough to cause any obvious signs of acute toxicity. It is obvious that the maximum allowable concen­ tration of cadmium In drinking water would have to be much below the h9 level necessary to cause erne©is end, also, probably below the level necessary to cause the appearance of a yellow ring on the teeth. However, frcm the resalts of the analyses of tissues for cadmium it can be seen that the element is deposited in liver and kidney, even on the lowest c 9* Friherg, 1* J. of lad* %g* Tcadteol* JO, 32 (1948)* 10* legge, t* M* Am* lap. Chief Inspect* Factories for 1932* H. M* Stationery Office, 1921*, p* 71** 11* Cotter, 1* 1* and Cotter, B* 1* Arch# Xnd. 0yg. and Occupational Med* J, 1*95 (1951) * 12* Prtnei, F* J* Ind* %g* Tcecieol* 29, 315 (1947)* 13* Cangelosl, J* T* 0* S. toral Had* J* Jg, 408 (1941). It. Garter, J# S* Ball# 0# S* A w Medical Sept* f, 349 (1946). 15. Forstner, 0* E. Chemistry and Industry* 499 (1948)* 16. S. ther^. 21, 59 (1923). 25. Koohmam, M. and Groaven, C. Dent. med. Wochsehr. gi, it27 (1925). 26. Waltnm, L. S. 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