- Email: [email protected]
Brominated maize oil. II. Storage of lipid-bound bromine in pigs fed brominated maize oil
Fd Cosmet. ToxicoL Vol. 9, pp. 13-19. Pergamon Press 1971. Printed in Great Britain
Brominated Maize Oil. 11. Storage of Lipid-bound Bromine in Pigs Fed Brominated Maize Oil I. F. GAUNT--Toxicology S. D. GANGOLLI
Analytical Chemistry and
R. F. CRAMPTON
British Industrial Biological Research Association, Woodmansterne Road, Carshalton, Surrey, England (Received 17 September 1970) Ahstract--~Groups of two miniature pigs were fed brominated maize oil (BMO) at 100 or 400 mg/kg/day for 42 days. Analysis of tissues showed that lipid-bound bro ,mine was present in adipose tissue, liver, spleen, adrenal glands, brain, kidneys and lymph nodes~(l'he highest concentrations were in the spleen and adrenal glands, and no differences were apparent between the concentrations in each tissue at the two dose levels. Organic bromine was not detected in serum or urine. Single pigs were given 0, 20, 40, 80 or 160 mg BMO/kg/day for 64 days followed by a period of 109-178 days on a diet free of added BMO. The concentration of lipid-bound bromine in subcutaneous adipose tissue was followed using biopsy samples. It was found that these concentrations were related to the intake level and increased rapidly for the first 12-20 days, after which they rose more slowly. After the animals were returned to the control diet the concentration of lipid-bound bromine decreased at a rate which could be accounted for by dilution with newly-deposited fat. All the organs examined at autopsy, with the exception of the kidneys of the pig on the lowest dosage level, contained lipid-bound bromine. Histological examination showed no abnormalities in pigs fed 100 or 400 mg BMO/kg daily for 42 days or in pigs fed 20, 40 or 80 mg BMO/kg daily for 64 days followed by 144-178 days feeding with diet free of added BMO. In the pig fed 160 mg BMO/kg dally for 64 days and control diet for 109 days the lymph nodes examined contained substantial amounts of intracellular fat. INTRODUCTION The current legislative p o s i t i o n relating to the use o f b r o m i n a t e d vegetable oils in soft drinks a n d the d a t a available on the toxicity o f c o m p o u n d s o f this type were reviewed b y G a u n t , G r a s s o & G a n g o l l i (1971) in an i n t r o d u c t i o n to the r e p o r t o f their own study o n the short-term effects o f feeding b r o m i n a t e d maize oil ( B M O ) to rats. These a u t h o r s were unable to establish a no-effect level for B M O in rats, since dose-related tissue storage o f l i p i d - b o u n d b r o m i n e and fatty infiltration o f the liver occurred at all the dietary levels tested (0"05-0-8~o, a p p r o x i m a t e l y equivalent to 25-400 m g B M O / k g / d a y ) . Some fat d e p o s i t i o n occurred also in the kidneys a n d h e a r t at the higher levels o f a d m i n i s t r a t i o n . A d d i t i o n a l studies by G a u n t et aL (1971) indicated t h a t the b r o m i n a t e d lipid deposited in fatty tissue was n o t readily available for metabolism. 13
14
I . F . GAUNT, S. D. GANGOLLI
and
R. F. CRAMPTON
The studies in miniature pigs reported here were undertaken to investigate further the dynamics of bromine storage following ingestion of BMO and to provide data on a nonrodent species. EXPE~MENTAL
Materials. The sample of BMO described previously (Gaunt et aL 1971) was used. Animals and diet. Miniature pigs of the Pitman-Moore crossed Palouse strain bred in these laboratories were given a basal diet of Hilean Rearers Pencils (British Oil and Cake Mills Ltd., London) and water ad lib. Experimental design and conduct Experiment 1. Two groups each of two female pigs (6-8 kg body weight) were given either 100 or 400 mg BMO/kg body weight each day for 42 days. BMO was mixed with a small quantity of powdered basal diet and Golden Syrup (Tate & Lyle Ltd., London) and was given before the main feed to ensure that the whole dose was eaten. After 42 days the pigs were killed and samples of urine, serum, perirenal adipose tissue, liver, spleen, adrenals, brain, kidneys and lymph nodes were taken for estimation of lipid-bound bromine. Tissues were preserved in 10~o buffered formalin and processed in the usual way for paraffin embedding. Sections were stained with haematoxylin and eosin for microscopic examination. Similar terminal studies were made on a control pig from the breeding colony. Experiment 2. Five pigs (two females and three males weighing 6-7 kg) were used. One pig was fed at each dose level of 0 (control), 20, 40, 80 and 160 mg/kg/day for 64 days, after which all pigs were fed the basal diet free of added BMO. The pigs were weighed daily. Samples of subcutaneous adipose tissue were taken from the flank areas under ether anaesthesia at intervals between days 5 and 57 and days 85 and 242, for estimation of lipid-bound bromine. The animals were killed between days 173 and 242 from the start of the test, and samples of heart, liver, kidneys, adrenals, thyroid, spleen, gonads, lung, brain, pancreas, lymph nodes, stomach, small intestine, colon, aorta and subcutaneous and perirenal adipose tissue were taken for determination of lipid-bound bromine concentrations and for histological examination. Known weights of each tissue were ground with sand, dried and extracted with diethyl ether in Soxhlet extractors. The fat contents were estimated gravimetrically after evaporation of the solvent. The lipid-bound bromine concentrations were estimated by saponifying the fats with 5 N alcoholic sodium hydroxide, ashing at 500°C, extracting the residues with water and estimating the bromine in this extract by the method of Hunter (1955). RESULTS Experiment 1 After pigs had been fed BMO for 42 days, lipid-bound bromine was present in all the tissues analysed (Table I), the concentrations being similar whether the pigs were fed 100 mg BMO/kg/day or four times this quantity. No organic bromine was detected in urine or serum. No abnormalities were seen in the histopathological examination.
Experiment 2 Body weights recorded during the periods of BMO and basal-diet feeding are shown in Table 2. It is difficult to assess the significance of weight gain in single animals of different
LIPID-BOUND BROMINEFROM BROMINATEDOIL
15
Table 1. Concentration of lipid-bound bromine in the tissues of pigs fed O, 100 or 400 rag BMO/kg[day for 42 days
Lipid-bound bromine content (mg/100 g fat)* Tissue or body fluid
BMO dosage (mg/kg/day) .... 100
400
880 650 4500 3800 2700 2500 1600 ND ND
910 650 4700 3800 2500 2400 1900 ND ND
Perirenal adipose tissue Liver Spleen Adrenals Brain Kidneys Lymph nodes Serum Urine
ND ----None detected * Values are means for groups of two animals. No lipid-bound bromine was detected in any of the same selection of tissues taken from one control pig fed the basal diet without added BMO. sexes but compared with the other two males the pig fed 160 mg BMO/kg/day gained weight at a slower rate from day 30 onwards and at autopsy was 13~o lighter. At all levels of BMO treatment, lipid-bound bromine was present in the subcutaneous adipose tissue generally from day 5 (Table 3), and there was a 12-20-day period of rapid increase in concentration followed by a slower rate of increase. The concentrations were dose-related. When pigs were returned to a diet containing no added bromine the concentrations gradually decreased but after 173 days bromine was still present in the subcutaneous adipose tissue. Lipid-bound bromine was also present in all the tissues of BMO-treated pigs killed after consuming a control diet for 109-178 days, with the exception of kidneys from the pig at the lowest dose level (Table 4). There were no differences in the appearance, weight or fat content of the organs of control and BMO-treated pigs. The only abnormality seen in the microscopic examination was deposition of fat in the lymph nodes of the pig fed 160 mg BMO/kg/day. DISCUSSION Lipid-bound bromine was present in all of the organs examined from pigs fed for 42 days on BMO at 100 or 400 mg/kg/day. The highest bromine concentration (4-5 g bromine/100 g fat) occurred in fat extracted from the spleen. One of the lowest concentrations was found in adipose tissue, which contained approximately 0-9 g bromine/100 g fat. Serial biopsy of subcutaneous adipose tissue from pigs fed 0, 20, 40, 80 or 160 mg BMO/kg body weight showed that there was a rapid rise in the concentration of lipid-bound bromine during the first 12-20 days, after which the concentration rose more slowly. This pattern is similar to that in rats fed approximately 400 mg/kg/day for up to 25 wk (Gaunt et al. 1971). The effect of returning animals to a diet containing no added BMO after a period on BMO treatment was also similar in the rat and pig. There was a gradual decrease in bromine
16
Io F. GAUNT, S. D. GANGOLLI a I l d R. F. CRAMPTON
t~ .q. t-4
4-o~ ¢'4
" 6t '~~ 6o o" e.
t'~ r~
e, °O ~
O u
II
O
.D
e)
t 'q q~
tt~
O
t,i
E~
e~ K
.Z *O
•
+.-~
LIPID-BOUND BROMINE FROM BROMINATED OIL
17
Table 4. Lipid-bound bromine concentrations in the fat of organs of pigs fed 0-160 mg BMO/kg/day for 64 days followed by a diet free of added BMO Lipid-bound bromine concentration (mg/100 g fat) BMO dosage (mg/kg/day) . . . . Organ Spleen Kidneys Liver Stomach Lung Colon Small intestine Brain Pancreas Heart Skeletal muscle Perirenal adipose tissue Lymph nodes Aorta Adrenals Thyroid Ovaries Testes
Days on BMO-free diet . . . .
0
20
40
80
160
242
178
178
144
109
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND --
16.5 ND 7.9 21 9.5 0.3 12.2 4.9 0.6 2.4 10.8 1.2 5-1 6.6 20 5-1 -4.5
29 2.2 17.2 35 17-8 1-1 12-5 5"4 0.9 9-5 15.1 2-3 5-9 8.1 39 13"0 -6"4
158 2-5 116 41 97 1.3 12.1 38 1.6 6.5 14.5 19.3 3.0 99 86 6-1 41 --
1585 202 436 42 1640 55 76 82 2820 325 350 40 93 495 200 146 -62
ND = None detected Values are for samples from one pig at each dose level.
c o n c e n t r a t i o n in adipose-tissue lipid. In the r a t it was suggested t h a t the decrease was due to dilution o f the organic b r o m i n e by the fat laid d o w n d u r i n g growth. In the pig a 50yo decrease in the c o n c e n t r a t i o n o f b r o m i n e in adipose tissue occurred in 48-71 days. D u r i n g this p e r i o d the b o d y weight increased by 60-100~o showing t h a t the decrease in concentration was consistent with dilution in new b o d y tissue. This suggests t h a t if l i p i d - b o u n d b r o mine is metabolized, the m e t a b o l i s m occurs only at a slow rate. Bromine was f o u n d in the fat o f all tissues (except in kidney at the lowest level o f B M O treatment) when the animals were a u t o p s i e d 109-178 days after r e t u r n i n g to the c o n t r o l diet. The highest concentrations were f o u n d in the fat o f pancreas, lung, spleen, aorta, liver, skeletal muscle a n d heart, with lower concentrations in o t h e r organs. The chemical f o r m in which the b r o m i n e occurs in the lipid is n o t known. F u r t h e r structural analysis w o u l d be necessary to determine whether it is present in the original form, as fed, o r in some o t h e r f o r m resulting f r o m resynthesis o f the m e t a b o l i t e s o f the b r o m i n a t e d fatty acids. D e p o s i t i o n o f fat was shown histologically in the liver a n d kidney o f rats fed a diet supplying a p p r o x i m a t e l y 100 o r 400 m g B M O / k g / d a y for 90 days ( G a u n t et aL 1971) o r fed b r o m i n a t e d cottonseed oil at a b o u t 250 m g / k g / d a y for 80 days ( M u n r o , M i d d l e t o n & Grice, 1969). N o deposition o f fat was seen in these organs in pigs fed for 42 days at the same levels, or in pigs fed for 64 days at lower levels a n d afterwards o n the c o n t r o l diet. However, l y m p h nodes f r o m the pig on the 160 m g / k g level contained large deposits o f rOOD 9 / I - - B
18
I.F. GAUNT, S. D. GANGOLLIa n d R. F. CRAMPTON
intracellular lipid 109 days after the last dose of BMO. Chemical analysis of these nodes showed that the lipid contained 93 mg bromine/100 g. It is concluded from the present study and from that in rats ( G a u n t et aL 1971) that daily intakes as low as 20--25 mg B M O / k g body weight allow a c c u m u l a t i o n of l i p i d - b o u n d b r o m i n e t h r o u g h o u t the fatty tissues of animals. Attempts to remove b r o m i n e by feeding BMO-treated animals on a diet free of added B M O for varying periods and by accelerating fatty tissue b r e a k d o w n by reducing the food intake, failed to show conclusively that b r o m i n e is mobilized from the tissues and metabolized. F u r t h e r investigation to establish the a m o u n t s of B M O that can be absorbed from the gastro-intestinal tract a n d the rate at which this organic b r o m i n e can be eliminated would be necessary to determine the level of intake of this substance that is without risk of tissue accumulation. Acknowledgements--The authors are grateful to Dr. P. Grasso and Dr. A. B. G. Lansdown for examining the histological slides, to the staff of the BIBRA Animal House for maintainingthe animals used and to the staff of the Toxicology and Analytical Chemistry Departments for technical assistance.
REFERENCES Gaunt, I. F., Grasso, P. & Gangolli, S. D. (1971). Brominated maize oil. I. Short-term toxicity and brominestorage studies in rats fed brominated maize oil. Fd Cosmet. ToxicoL 9, 1. Hunter, G. (1955). Micro-determination of bromide in body fluids. Biocbem. J. 60, 261. Munro, I. C., Middleton, E. J. & Grice, H. C. (1969). Biochemical and pathological changes in rats fed brominated cottonseed oil for 80 days. Fd Cosmet. ToxicoL 7, 25.
Recherches sur l'huile de mais bromur~e. II. L'accnmulation du brome fix~ par les lipides chez les pores nourris d'huile de mais bromm'~e Rth'um~---Des groupes de deux pores miniatures ont eonsomm6 pendant 42 jours 100 ou 400 mg/kg/jour d'huile de mats bromur6e (HMB). L'analyse des tissus a r6v616la pr6sence de brome fuc6par les lipides darts le tissu adipeux, le foie, la rate, ies surr6nales, le cerveau, les reins et les nodules lymphatiques. Les concentrations les plus fortes ont 6t6 trouv6es dam la rate et darts les surr6nales et aucune ditfdrence ne pouvait ~tre observ~ entre les concentrations darts chaque tissu, pour les deux dosages. On n'a pas d6c.el6de brome organique darts le s6rum et dans l'urine. Des pores ont consomm6, individuellement, 0, 20, 40, 80 ou 160 mg d'HMB/kg/jour pendant 64 jours puis, pendant une p~riode de 109 ~ 178jours, des rations exemptes d'HMB. L'6volution des concentrations de brome fix6 par les lipides dans le tissu adipeux sous-cutan6 a 6t6 suivie sur des pr61~vementsbiopsiques. On a constat6 ainsi que ces concentrations 6taient en corr61ation avec le niveau des ingesta, augmentaient rapidement pendant les 12 ~ 20 premiers jours, puis plus lentement. Le retour au r6gime t6moin a entrain6 une diminution du taux de brome fix6 par les lipides, ceei/t une vitesse explicable par la dilution dans de nouveaux d6p6ts de graisse. A l'exception des reins du pore qui recevait la dose la plus faible, tousles organes examines ~ rautopsie contenaient du brome fix6 par les lipides. L'examen histologique n'a r6v616aueune anomalie chez lenspores qui avaient consomm6 pendant 42jours 100 ou 400 mg I-IMB/kg/jour ni chez les pores qui en avaient consomm6 20, 40 ou 80 mg/kg/jour pendant 64 jours, puis avaient 6t6 transf6r~s pendant 144 h 178 jours an r6gime sans HMB. Des quantit~s substantielles de graisse intracellulaire ont 6t6 trouv6es darts les nodules lymphatiques du pore qui avait consomm6 pendant 64 jours 160 mg I-IMB/kg/jour puis, pendant 109 jours, la ration t6moin.
LIPID-BOUND BROMINE FROM BROMINATFD OIL Bromiertes Mais61. H. Speicherung yon lipidgebundenem Brom in Schweinen, an die bromiertes Mais61 verffittert wurde Zusammenfassung--An Gruppen yon je zwei Zwergschweinen wurde bromiertes MaisSl (BMO) in Dosen yon 100 oder 400 mg/kg/Tag 42 Tage fang verfflttert. Die Analyse der Geweb¢ zeigte, dass lipidgebundenes Brom im Fettgewebe, in der Leber, Milz, Nebennieren, Gehim, Nieren und Lymphknoten vorhanden war. Die h~chsten Konzentrationen warden in Milz und Nebennieren festgestellt, und es wurden keine Unterschiede zwischen den Konzentrationen in jedem Gewebe bei den zwei Dosierungen gefunden. Organisches Brom wurd¢ im Serum oder Urin nicht festgestellt. Einzelne Schweine erhielten 0, 20, 40, 80 oder 160 mg BMO/kg/Tag ffu" die Dauer yon 64 Tagen, woran sich eine Periode von 109-178 Tagen mit Futter ohne BMO-Zusatz ansehloss. Die Konzentration yon lipidgebundenem Brom im subcutanen Fettgewebe wurdo mittels Probeexzisionen festgesteIlt. Es zeigte sich, dass diese Konzentrationen zur Aufnahmekonzentration in Beziehung standen und in den ersten 12-20 Tagen rasch zunahmen, w~u'end der weitere Anstieg langsamer erfolgte. Nach der Rfickkehr zum Kontrollfutter nahm die Konzentration yon lipidgebundenem Brom mit einer Geschwindigkeit at), die sich mit der Verdtinnung dutch neuangelagertes Fett erklSxen liess. Alle bei der Sektion untersuchten Organe mit Ausnahme der Nieren des Schweins, das die geringste Dosierung erhalten hatte, enthielten lipidgebundenes Brom. Die histologische Untersuchung zeigte keine Anomalien bei Schweinen, die 100 oder 400 mg BMO/kg ttiglich 42 Tage lang, und bei Schweinen, die 20, 40 oder 80 mg BMO/kg t~glich 64 Tage lang, und anschliessend 144-178 Tage lang ein Futter ohne Zusatz yon BMO erhalten hatten. Bei dem Schwein, das 160 mg BMO/kg tiglich 64 Tage lang und 109 Tage lang Kontrolffutter erhalten hatte, enthielten die untersuchten Lymphknoten wesentlich¢ Mengen intracellul~en Fetts.
19
Brominated Maize Oil. 11. Storage of Lipid-bound Bromine in Pigs Fed Brominated Maize Oil I. F. GAUNT--Toxicology S. D. GANGOLLI
Analytical Chemistry and
R. F. CRAMPTON
British Industrial Biological Research Association, Woodmansterne Road, Carshalton, Surrey, England (Received 17 September 1970) Ahstract--~Groups of two miniature pigs were fed brominated maize oil (BMO) at 100 or 400 mg/kg/day for 42 days. Analysis of tissues showed that lipid-bound bro ,mine was present in adipose tissue, liver, spleen, adrenal glands, brain, kidneys and lymph nodes~(l'he highest concentrations were in the spleen and adrenal glands, and no differences were apparent between the concentrations in each tissue at the two dose levels. Organic bromine was not detected in serum or urine. Single pigs were given 0, 20, 40, 80 or 160 mg BMO/kg/day for 64 days followed by a period of 109-178 days on a diet free of added BMO. The concentration of lipid-bound bromine in subcutaneous adipose tissue was followed using biopsy samples. It was found that these concentrations were related to the intake level and increased rapidly for the first 12-20 days, after which they rose more slowly. After the animals were returned to the control diet the concentration of lipid-bound bromine decreased at a rate which could be accounted for by dilution with newly-deposited fat. All the organs examined at autopsy, with the exception of the kidneys of the pig on the lowest dosage level, contained lipid-bound bromine. Histological examination showed no abnormalities in pigs fed 100 or 400 mg BMO/kg daily for 42 days or in pigs fed 20, 40 or 80 mg BMO/kg daily for 64 days followed by 144-178 days feeding with diet free of added BMO. In the pig fed 160 mg BMO/kg dally for 64 days and control diet for 109 days the lymph nodes examined contained substantial amounts of intracellular fat. INTRODUCTION The current legislative p o s i t i o n relating to the use o f b r o m i n a t e d vegetable oils in soft drinks a n d the d a t a available on the toxicity o f c o m p o u n d s o f this type were reviewed b y G a u n t , G r a s s o & G a n g o l l i (1971) in an i n t r o d u c t i o n to the r e p o r t o f their own study o n the short-term effects o f feeding b r o m i n a t e d maize oil ( B M O ) to rats. These a u t h o r s were unable to establish a no-effect level for B M O in rats, since dose-related tissue storage o f l i p i d - b o u n d b r o m i n e and fatty infiltration o f the liver occurred at all the dietary levels tested (0"05-0-8~o, a p p r o x i m a t e l y equivalent to 25-400 m g B M O / k g / d a y ) . Some fat d e p o s i t i o n occurred also in the kidneys a n d h e a r t at the higher levels o f a d m i n i s t r a t i o n . A d d i t i o n a l studies by G a u n t et aL (1971) indicated t h a t the b r o m i n a t e d lipid deposited in fatty tissue was n o t readily available for metabolism. 13
14
I . F . GAUNT, S. D. GANGOLLI
and
R. F. CRAMPTON
The studies in miniature pigs reported here were undertaken to investigate further the dynamics of bromine storage following ingestion of BMO and to provide data on a nonrodent species. EXPE~MENTAL
Materials. The sample of BMO described previously (Gaunt et aL 1971) was used. Animals and diet. Miniature pigs of the Pitman-Moore crossed Palouse strain bred in these laboratories were given a basal diet of Hilean Rearers Pencils (British Oil and Cake Mills Ltd., London) and water ad lib. Experimental design and conduct Experiment 1. Two groups each of two female pigs (6-8 kg body weight) were given either 100 or 400 mg BMO/kg body weight each day for 42 days. BMO was mixed with a small quantity of powdered basal diet and Golden Syrup (Tate & Lyle Ltd., London) and was given before the main feed to ensure that the whole dose was eaten. After 42 days the pigs were killed and samples of urine, serum, perirenal adipose tissue, liver, spleen, adrenals, brain, kidneys and lymph nodes were taken for estimation of lipid-bound bromine. Tissues were preserved in 10~o buffered formalin and processed in the usual way for paraffin embedding. Sections were stained with haematoxylin and eosin for microscopic examination. Similar terminal studies were made on a control pig from the breeding colony. Experiment 2. Five pigs (two females and three males weighing 6-7 kg) were used. One pig was fed at each dose level of 0 (control), 20, 40, 80 and 160 mg/kg/day for 64 days, after which all pigs were fed the basal diet free of added BMO. The pigs were weighed daily. Samples of subcutaneous adipose tissue were taken from the flank areas under ether anaesthesia at intervals between days 5 and 57 and days 85 and 242, for estimation of lipid-bound bromine. The animals were killed between days 173 and 242 from the start of the test, and samples of heart, liver, kidneys, adrenals, thyroid, spleen, gonads, lung, brain, pancreas, lymph nodes, stomach, small intestine, colon, aorta and subcutaneous and perirenal adipose tissue were taken for determination of lipid-bound bromine concentrations and for histological examination. Known weights of each tissue were ground with sand, dried and extracted with diethyl ether in Soxhlet extractors. The fat contents were estimated gravimetrically after evaporation of the solvent. The lipid-bound bromine concentrations were estimated by saponifying the fats with 5 N alcoholic sodium hydroxide, ashing at 500°C, extracting the residues with water and estimating the bromine in this extract by the method of Hunter (1955). RESULTS Experiment 1 After pigs had been fed BMO for 42 days, lipid-bound bromine was present in all the tissues analysed (Table I), the concentrations being similar whether the pigs were fed 100 mg BMO/kg/day or four times this quantity. No organic bromine was detected in urine or serum. No abnormalities were seen in the histopathological examination.
Experiment 2 Body weights recorded during the periods of BMO and basal-diet feeding are shown in Table 2. It is difficult to assess the significance of weight gain in single animals of different
LIPID-BOUND BROMINEFROM BROMINATEDOIL
15
Table 1. Concentration of lipid-bound bromine in the tissues of pigs fed O, 100 or 400 rag BMO/kg[day for 42 days
Lipid-bound bromine content (mg/100 g fat)* Tissue or body fluid
BMO dosage (mg/kg/day) .... 100
400
880 650 4500 3800 2700 2500 1600 ND ND
910 650 4700 3800 2500 2400 1900 ND ND
Perirenal adipose tissue Liver Spleen Adrenals Brain Kidneys Lymph nodes Serum Urine
ND ----None detected * Values are means for groups of two animals. No lipid-bound bromine was detected in any of the same selection of tissues taken from one control pig fed the basal diet without added BMO. sexes but compared with the other two males the pig fed 160 mg BMO/kg/day gained weight at a slower rate from day 30 onwards and at autopsy was 13~o lighter. At all levels of BMO treatment, lipid-bound bromine was present in the subcutaneous adipose tissue generally from day 5 (Table 3), and there was a 12-20-day period of rapid increase in concentration followed by a slower rate of increase. The concentrations were dose-related. When pigs were returned to a diet containing no added bromine the concentrations gradually decreased but after 173 days bromine was still present in the subcutaneous adipose tissue. Lipid-bound bromine was also present in all the tissues of BMO-treated pigs killed after consuming a control diet for 109-178 days, with the exception of kidneys from the pig at the lowest dose level (Table 4). There were no differences in the appearance, weight or fat content of the organs of control and BMO-treated pigs. The only abnormality seen in the microscopic examination was deposition of fat in the lymph nodes of the pig fed 160 mg BMO/kg/day. DISCUSSION Lipid-bound bromine was present in all of the organs examined from pigs fed for 42 days on BMO at 100 or 400 mg/kg/day. The highest bromine concentration (4-5 g bromine/100 g fat) occurred in fat extracted from the spleen. One of the lowest concentrations was found in adipose tissue, which contained approximately 0-9 g bromine/100 g fat. Serial biopsy of subcutaneous adipose tissue from pigs fed 0, 20, 40, 80 or 160 mg BMO/kg body weight showed that there was a rapid rise in the concentration of lipid-bound bromine during the first 12-20 days, after which the concentration rose more slowly. This pattern is similar to that in rats fed approximately 400 mg/kg/day for up to 25 wk (Gaunt et al. 1971). The effect of returning animals to a diet containing no added BMO after a period on BMO treatment was also similar in the rat and pig. There was a gradual decrease in bromine
16
Io F. GAUNT, S. D. GANGOLLI a I l d R. F. CRAMPTON
t~ .q. t-4
4-o~ ¢'4
" 6t '~~ 6o o" e.
t'~ r~
e, °O ~
O u
II
O
.D
e)
t 'q q~
tt~
O
t,i
E~
e~ K
.Z *O
•
+.-~
LIPID-BOUND BROMINE FROM BROMINATED OIL
17
Table 4. Lipid-bound bromine concentrations in the fat of organs of pigs fed 0-160 mg BMO/kg/day for 64 days followed by a diet free of added BMO Lipid-bound bromine concentration (mg/100 g fat) BMO dosage (mg/kg/day) . . . . Organ Spleen Kidneys Liver Stomach Lung Colon Small intestine Brain Pancreas Heart Skeletal muscle Perirenal adipose tissue Lymph nodes Aorta Adrenals Thyroid Ovaries Testes
Days on BMO-free diet . . . .
0
20
40
80
160
242
178
178
144
109
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND --
16.5 ND 7.9 21 9.5 0.3 12.2 4.9 0.6 2.4 10.8 1.2 5-1 6.6 20 5-1 -4.5
29 2.2 17.2 35 17-8 1-1 12-5 5"4 0.9 9-5 15.1 2-3 5-9 8.1 39 13"0 -6"4
158 2-5 116 41 97 1.3 12.1 38 1.6 6.5 14.5 19.3 3.0 99 86 6-1 41 --
1585 202 436 42 1640 55 76 82 2820 325 350 40 93 495 200 146 -62
ND = None detected Values are for samples from one pig at each dose level.
c o n c e n t r a t i o n in adipose-tissue lipid. In the r a t it was suggested t h a t the decrease was due to dilution o f the organic b r o m i n e by the fat laid d o w n d u r i n g growth. In the pig a 50yo decrease in the c o n c e n t r a t i o n o f b r o m i n e in adipose tissue occurred in 48-71 days. D u r i n g this p e r i o d the b o d y weight increased by 60-100~o showing t h a t the decrease in concentration was consistent with dilution in new b o d y tissue. This suggests t h a t if l i p i d - b o u n d b r o mine is metabolized, the m e t a b o l i s m occurs only at a slow rate. Bromine was f o u n d in the fat o f all tissues (except in kidney at the lowest level o f B M O treatment) when the animals were a u t o p s i e d 109-178 days after r e t u r n i n g to the c o n t r o l diet. The highest concentrations were f o u n d in the fat o f pancreas, lung, spleen, aorta, liver, skeletal muscle a n d heart, with lower concentrations in o t h e r organs. The chemical f o r m in which the b r o m i n e occurs in the lipid is n o t known. F u r t h e r structural analysis w o u l d be necessary to determine whether it is present in the original form, as fed, o r in some o t h e r f o r m resulting f r o m resynthesis o f the m e t a b o l i t e s o f the b r o m i n a t e d fatty acids. D e p o s i t i o n o f fat was shown histologically in the liver a n d kidney o f rats fed a diet supplying a p p r o x i m a t e l y 100 o r 400 m g B M O / k g / d a y for 90 days ( G a u n t et aL 1971) o r fed b r o m i n a t e d cottonseed oil at a b o u t 250 m g / k g / d a y for 80 days ( M u n r o , M i d d l e t o n & Grice, 1969). N o deposition o f fat was seen in these organs in pigs fed for 42 days at the same levels, or in pigs fed for 64 days at lower levels a n d afterwards o n the c o n t r o l diet. However, l y m p h nodes f r o m the pig on the 160 m g / k g level contained large deposits o f rOOD 9 / I - - B
18
I.F. GAUNT, S. D. GANGOLLIa n d R. F. CRAMPTON
intracellular lipid 109 days after the last dose of BMO. Chemical analysis of these nodes showed that the lipid contained 93 mg bromine/100 g. It is concluded from the present study and from that in rats ( G a u n t et aL 1971) that daily intakes as low as 20--25 mg B M O / k g body weight allow a c c u m u l a t i o n of l i p i d - b o u n d b r o m i n e t h r o u g h o u t the fatty tissues of animals. Attempts to remove b r o m i n e by feeding BMO-treated animals on a diet free of added B M O for varying periods and by accelerating fatty tissue b r e a k d o w n by reducing the food intake, failed to show conclusively that b r o m i n e is mobilized from the tissues and metabolized. F u r t h e r investigation to establish the a m o u n t s of B M O that can be absorbed from the gastro-intestinal tract a n d the rate at which this organic b r o m i n e can be eliminated would be necessary to determine the level of intake of this substance that is without risk of tissue accumulation. Acknowledgements--The authors are grateful to Dr. P. Grasso and Dr. A. B. G. Lansdown for examining the histological slides, to the staff of the BIBRA Animal House for maintainingthe animals used and to the staff of the Toxicology and Analytical Chemistry Departments for technical assistance.
REFERENCES Gaunt, I. F., Grasso, P. & Gangolli, S. D. (1971). Brominated maize oil. I. Short-term toxicity and brominestorage studies in rats fed brominated maize oil. Fd Cosmet. ToxicoL 9, 1. Hunter, G. (1955). Micro-determination of bromide in body fluids. Biocbem. J. 60, 261. Munro, I. C., Middleton, E. J. & Grice, H. C. (1969). Biochemical and pathological changes in rats fed brominated cottonseed oil for 80 days. Fd Cosmet. ToxicoL 7, 25.
Recherches sur l'huile de mais bromur~e. II. L'accnmulation du brome fix~ par les lipides chez les pores nourris d'huile de mais bromm'~e Rth'um~---Des groupes de deux pores miniatures ont eonsomm6 pendant 42 jours 100 ou 400 mg/kg/jour d'huile de mats bromur6e (HMB). L'analyse des tissus a r6v616la pr6sence de brome fuc6par les lipides darts le tissu adipeux, le foie, la rate, ies surr6nales, le cerveau, les reins et les nodules lymphatiques. Les concentrations les plus fortes ont 6t6 trouv6es dam la rate et darts les surr6nales et aucune ditfdrence ne pouvait ~tre observ~ entre les concentrations darts chaque tissu, pour les deux dosages. On n'a pas d6c.el6de brome organique darts le s6rum et dans l'urine. Des pores ont consomm6, individuellement, 0, 20, 40, 80 ou 160 mg d'HMB/kg/jour pendant 64 jours puis, pendant une p~riode de 109 ~ 178jours, des rations exemptes d'HMB. L'6volution des concentrations de brome fix6 par les lipides dans le tissu adipeux sous-cutan6 a 6t6 suivie sur des pr61~vementsbiopsiques. On a constat6 ainsi que ces concentrations 6taient en corr61ation avec le niveau des ingesta, augmentaient rapidement pendant les 12 ~ 20 premiers jours, puis plus lentement. Le retour au r6gime t6moin a entrain6 une diminution du taux de brome fix6 par les lipides, ceei/t une vitesse explicable par la dilution dans de nouveaux d6p6ts de graisse. A l'exception des reins du pore qui recevait la dose la plus faible, tousles organes examines ~ rautopsie contenaient du brome fix6 par les lipides. L'examen histologique n'a r6v616aueune anomalie chez lenspores qui avaient consomm6 pendant 42jours 100 ou 400 mg I-IMB/kg/jour ni chez les pores qui en avaient consomm6 20, 40 ou 80 mg/kg/jour pendant 64 jours, puis avaient 6t6 transf6r~s pendant 144 h 178 jours an r6gime sans HMB. Des quantit~s substantielles de graisse intracellulaire ont 6t6 trouv6es darts les nodules lymphatiques du pore qui avait consomm6 pendant 64 jours 160 mg I-IMB/kg/jour puis, pendant 109 jours, la ration t6moin.
LIPID-BOUND BROMINE FROM BROMINATFD OIL Bromiertes Mais61. H. Speicherung yon lipidgebundenem Brom in Schweinen, an die bromiertes Mais61 verffittert wurde Zusammenfassung--An Gruppen yon je zwei Zwergschweinen wurde bromiertes MaisSl (BMO) in Dosen yon 100 oder 400 mg/kg/Tag 42 Tage fang verfflttert. Die Analyse der Geweb¢ zeigte, dass lipidgebundenes Brom im Fettgewebe, in der Leber, Milz, Nebennieren, Gehim, Nieren und Lymphknoten vorhanden war. Die h~chsten Konzentrationen warden in Milz und Nebennieren festgestellt, und es wurden keine Unterschiede zwischen den Konzentrationen in jedem Gewebe bei den zwei Dosierungen gefunden. Organisches Brom wurd¢ im Serum oder Urin nicht festgestellt. Einzelne Schweine erhielten 0, 20, 40, 80 oder 160 mg BMO/kg/Tag ffu" die Dauer yon 64 Tagen, woran sich eine Periode von 109-178 Tagen mit Futter ohne BMO-Zusatz ansehloss. Die Konzentration yon lipidgebundenem Brom im subcutanen Fettgewebe wurdo mittels Probeexzisionen festgesteIlt. Es zeigte sich, dass diese Konzentrationen zur Aufnahmekonzentration in Beziehung standen und in den ersten 12-20 Tagen rasch zunahmen, w~u'end der weitere Anstieg langsamer erfolgte. Nach der Rfickkehr zum Kontrollfutter nahm die Konzentration yon lipidgebundenem Brom mit einer Geschwindigkeit at), die sich mit der Verdtinnung dutch neuangelagertes Fett erklSxen liess. Alle bei der Sektion untersuchten Organe mit Ausnahme der Nieren des Schweins, das die geringste Dosierung erhalten hatte, enthielten lipidgebundenes Brom. Die histologische Untersuchung zeigte keine Anomalien bei Schweinen, die 100 oder 400 mg BMO/kg ttiglich 42 Tage lang, und bei Schweinen, die 20, 40 oder 80 mg BMO/kg t~glich 64 Tage lang, und anschliessend 144-178 Tage lang ein Futter ohne Zusatz yon BMO erhalten hatten. Bei dem Schwein, das 160 mg BMO/kg tiglich 64 Tage lang und 109 Tage lang Kontrolffutter erhalten hatte, enthielten die untersuchten Lymphknoten wesentlich¢ Mengen intracellul~en Fetts.
19