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Biochemical aspects of toxicology of atromid
JOURNAL OF ATHEROSCLEROSIS RESEARCH
737
B I O C H E M I C A L ASPECTS OF TOXICOLOGY OF A T R O M I D
J. M. T H O R P
Imperial Chemical Industries Ltd., Pharmaceuticals Division, Alderley Park, Macclesfield, Cheshire (England)
The concept that disordered lipid metabolism is primarily centred in the liver implies that a treatment aimed at correcting this will affect liver metabolism. It is important that this should not be associated with any impairment of normal liver function. We therefore examined in detail the effects of Atromid treatment on liver function in experimental animals. In spite of measurable changes in the composition of the liver, consisting of an increase in protein and a reduction in lipids and glycogen, no alteration in liver excretory or detoxifying activity was found in the rat or monkey. Although we have found little, if any, change in reticulo-endothelial activity in our animals, OVERBEEK (personal communication) has reported that Atromid caused an increased clearance of a carbon suspension in ovariectomized rats. The interpretation of changes in serum and liver enzyme activity is to a large extent influenced by the circumstances under which these are normally observed; generally some pathological or toxic process. The changes in transaminases which have been observed in Atromid-treated animals appear to result from a true increase in the concentration of enzyme in the liver, which is maintained at a constant ratio to that in serum. Since changes in these and other enzymes can be induced b y fasting (or refeeding of fasting animals), adrenalectomy, and b y cortisol or thyroxine treatment, it is reasonable to assume that they reflect the general metabolic state of the animal, and of the liver in particular. In the extreme example of pyridoxine deficiency, which leads to a deficiency of transaminase activity, a fatty liver and atherosclerosis develops quite rapidly in experimental animals. Part of the apparent "pyridoxalsparing" effect of Atromid m a y be due to a redistribution of substrate or coenzyme between non-specific protein such as albumin, and the specific apo-enzyme. We have found that the activity of liver transaminase is reduced in the presence of albumin in vitro, and that activity can be restored by the prior addition of ethyl-a-(p-chlorophenoxy)-isobutyrate (CPIB) to the albumin, but not if added subsequently. Previously we had found 1 that two changes occurred in SGOT following high doses of Atromid in the monkey. The first consists of a transient rise in the SGOT immediately (2-4 h) after each dose (including the first), the second change is a gradual increase in SGOT in parallel with, and equivalent to, an increase in liver GOT activity. At effective doses in the rat CPIB is undetectable in liver tissue, and about 50-60 ~o of the compound excreted in urine is present as the glucuronide. The histological J. Atheroscler. Res., 3 (1963) 737-739
738
j.M. THORP
changes seen in rats treated with high doses of CPIB are less marked than those in animals treated with low doses of related compounds which are less efficiently conjugated with glucuronic acid. The efficiency of glucuronide conjugation of CPIB increases from t h e rat to monkey to man. Since there is no histological change in the liver of the monkey, if such a correlation is valid, little of the changes seen in the rat are likely to occur in man. Rabbit foetal serum has been found to accumulate higher concentrations of CPIB than those in the maternal serum. Since the foetus is devoid of the glucuronideconjugating enzyme required for excretion of CPIB, the use of Atromid in pregnancy is contraindicated on purely biochemical grounds. The effects of CPIB on endocrine function had also to be examined. Even with the addition of androsterone, no histological or weight change was evident in the testes and accessory glands of the male rat or monkey, or the sex organs of the female rat. The adrenals had a slightly higher content of cholesterol, and were somewhat lighter than the controls. This is compatible with a potentiation of the activity of endogenous steroids b y CPIB, as was observed with exogenous steroid hormones. No change in thyroid uptake of 1811 was found in CPIB-treated rats. It has been shown 2 that even though 2,4-dichloro-phenoxyacetic acid displaces protein-bound thyroxine into the liver of the rat, this is not accompanied b y any change in thyroid function, presumably because the non protein-bound effective concentration of thyroxine regulating thyrotrophin secretion is unaltered. In brief then, the actions of CPIB and therefore any possible toxic effects which m a y arise appear to be limited to an endocrine-mediated action on liver metabolism, and a competitive inhibition of renal tubular transport of acids. SUMMARY
The influence of Atromid on liver and serum enzyme activity, on the foetus, and on the endocrine system in experimental animals, is briefly discussed. Such changes as do occur are considered to reflect the intrinsic mode of action of Atromid, are modified by species differences, and do not result in any impairment of function. RESUMI~
L'influence qu'exerce l'Atromide sur l'activit6 enzymatique du foie et du sdrum, sur le foetus et sur le syst6me endocrin chez l'animal d'expdrimentation, est bri~vement discut6e. On estime que les changements qui surviennent, traduisent l'intrins~que mode d'action de l'Atromide, qu'ils diff6rent selon l'esp~ce et qu'~ls ne portent atteinte ~t aucune fonction. ZUSAMMENFASSUNG
Der Einfluss von Atromid auf Leber- und Serumenzymaktivit~t, auf das F6tus und auf das endokrine System bei Versuchstieren, wird kurz besprochen. Die auftretenden J. A theroscler. Res., 3 (1963) 737-739
BIOCHEMICAL ASPECTS OF TOXICOLOGY OF ATROMID
Ver~nderungen werden betrachtet den
als A u s s e r u n g e n d e r i n n e r e n W i r k u n g
modifiziert
nach
unterschiedlichen
Funktionen
nicht beeintr~chtigend.
untersuchten
Arten,
und
739
des Atromid, die n o r m a l e n
REFERENCES I j. M. THORP, Lancet, i (1962) 1323. 2 W. M. FLORSHEIM, S M. VELCOFF AND A. n . WILLIAMS, Endocrinology, 72 (1963) 327.
DISCUSSION K. G. GREEN
(CHAIRMAN)
OLIVER (Edinburgh) : I would like to place on record m y opinion t h a t A t r o m i d should never be given during pregnancy, unless for a very over-riding reason. Over 8 years ago G. S. BOYD and I, and again more recently, investigated s e r u m lipid levels in p r e g n a n t w o m e n and found a considerable increase in s e r u m cholesterol, phospholipid and triglyceride in the last trimester. This is a physiological response to n o r m a l p r e g n a n c y and it would be w r o n g to t r y to alter it. GRESHAM (Cambmdge): We have heard a great deal a b o u t the results of C P I B on the lipids in h u m a n s , b u t if we are aiming at finding out the mode of action of the drug, more pertinent information m i g h t be obtained from work in animals. There are several experimental models which could be used. The r a t fed on u n s a t u r a t e d f a t t y acids develops t h r o m b o s e s which are associated with raised s e r u m triglyceride levels. W h a t would C P I B or Atromid do to these? At 0.3 % Atromid in a high fat diet for four m o n t h s , we have been able to show no effect on the d e v e l o p m e n t of lesions in the aorta, t h o u g h there was a small b u t significant drop in the s e r u m cholesterol of these animals. I do not regard r a b b i t atherosclerosis as being very similar to the h u m a n condition, however, and I suggest t h a t e x p e r i m e n t s on platelet adhesiveness in animals on high fat diets, such as the e x p e r i m e n t s described by MUSTARD, m i g h t also be relevant. THORP (I C.I.) : We have in fact looked at w h a t h a p p e n s in the r a b b i t fed on a cholesterolsupplem e n t e d diet. Simultaneous a d m i n i s t r a t i o n of A t r o m l d to such animals does reduce the cholesterol level slightly. I t is n o t a very striking thing and I would imagine in the sort of e x p e r i m e n t t h a t DR. GRESHAM is describing the effect would be even smaller. Our experience in b o t h r a b b i t s and m o n k e y s has generally been t h a t the e x t e n t of aortic atherosclerosis associated w i t h these artificially elevated s e r u m lipids is almost directly proportional to the circulating level of lipids in life W h a t we h a v e done is to take r a b b i t s and to t r e a t t h e m for a period of 12 weeks with a cholesterol s u p p l e m e n t e d diet and m a t c h t h e m at the end of this period on the basis of their s e r u m lipids. Half were r e t u r n e d to a n o r m a l ' d i e t , and half p u t on a diet s u p p l e m e n t e d with A t r o m i d for 8 weeks. The rate of regression of the s e r u m lipids and the degree of atherosclerosis were measured. There was a small b u t significant reduction in aortic a t h e r o m a and some increase in the rate of fall of the s e r u m lipids in the treated group This I t h i n k only p a r t l y answers one of the questions which one hears asked continually: where does the cholesterol go? This particular e x p e r i m e n t suggests at least t h a t it does n o t go into the aorta.
J. Atheroscler. Res., 3 (1963) 737-739
737
B I O C H E M I C A L ASPECTS OF TOXICOLOGY OF A T R O M I D
J. M. T H O R P
Imperial Chemical Industries Ltd., Pharmaceuticals Division, Alderley Park, Macclesfield, Cheshire (England)
The concept that disordered lipid metabolism is primarily centred in the liver implies that a treatment aimed at correcting this will affect liver metabolism. It is important that this should not be associated with any impairment of normal liver function. We therefore examined in detail the effects of Atromid treatment on liver function in experimental animals. In spite of measurable changes in the composition of the liver, consisting of an increase in protein and a reduction in lipids and glycogen, no alteration in liver excretory or detoxifying activity was found in the rat or monkey. Although we have found little, if any, change in reticulo-endothelial activity in our animals, OVERBEEK (personal communication) has reported that Atromid caused an increased clearance of a carbon suspension in ovariectomized rats. The interpretation of changes in serum and liver enzyme activity is to a large extent influenced by the circumstances under which these are normally observed; generally some pathological or toxic process. The changes in transaminases which have been observed in Atromid-treated animals appear to result from a true increase in the concentration of enzyme in the liver, which is maintained at a constant ratio to that in serum. Since changes in these and other enzymes can be induced b y fasting (or refeeding of fasting animals), adrenalectomy, and b y cortisol or thyroxine treatment, it is reasonable to assume that they reflect the general metabolic state of the animal, and of the liver in particular. In the extreme example of pyridoxine deficiency, which leads to a deficiency of transaminase activity, a fatty liver and atherosclerosis develops quite rapidly in experimental animals. Part of the apparent "pyridoxalsparing" effect of Atromid m a y be due to a redistribution of substrate or coenzyme between non-specific protein such as albumin, and the specific apo-enzyme. We have found that the activity of liver transaminase is reduced in the presence of albumin in vitro, and that activity can be restored by the prior addition of ethyl-a-(p-chlorophenoxy)-isobutyrate (CPIB) to the albumin, but not if added subsequently. Previously we had found 1 that two changes occurred in SGOT following high doses of Atromid in the monkey. The first consists of a transient rise in the SGOT immediately (2-4 h) after each dose (including the first), the second change is a gradual increase in SGOT in parallel with, and equivalent to, an increase in liver GOT activity. At effective doses in the rat CPIB is undetectable in liver tissue, and about 50-60 ~o of the compound excreted in urine is present as the glucuronide. The histological J. Atheroscler. Res., 3 (1963) 737-739
738
j.M. THORP
changes seen in rats treated with high doses of CPIB are less marked than those in animals treated with low doses of related compounds which are less efficiently conjugated with glucuronic acid. The efficiency of glucuronide conjugation of CPIB increases from t h e rat to monkey to man. Since there is no histological change in the liver of the monkey, if such a correlation is valid, little of the changes seen in the rat are likely to occur in man. Rabbit foetal serum has been found to accumulate higher concentrations of CPIB than those in the maternal serum. Since the foetus is devoid of the glucuronideconjugating enzyme required for excretion of CPIB, the use of Atromid in pregnancy is contraindicated on purely biochemical grounds. The effects of CPIB on endocrine function had also to be examined. Even with the addition of androsterone, no histological or weight change was evident in the testes and accessory glands of the male rat or monkey, or the sex organs of the female rat. The adrenals had a slightly higher content of cholesterol, and were somewhat lighter than the controls. This is compatible with a potentiation of the activity of endogenous steroids b y CPIB, as was observed with exogenous steroid hormones. No change in thyroid uptake of 1811 was found in CPIB-treated rats. It has been shown 2 that even though 2,4-dichloro-phenoxyacetic acid displaces protein-bound thyroxine into the liver of the rat, this is not accompanied b y any change in thyroid function, presumably because the non protein-bound effective concentration of thyroxine regulating thyrotrophin secretion is unaltered. In brief then, the actions of CPIB and therefore any possible toxic effects which m a y arise appear to be limited to an endocrine-mediated action on liver metabolism, and a competitive inhibition of renal tubular transport of acids. SUMMARY
The influence of Atromid on liver and serum enzyme activity, on the foetus, and on the endocrine system in experimental animals, is briefly discussed. Such changes as do occur are considered to reflect the intrinsic mode of action of Atromid, are modified by species differences, and do not result in any impairment of function. RESUMI~
L'influence qu'exerce l'Atromide sur l'activit6 enzymatique du foie et du sdrum, sur le foetus et sur le syst6me endocrin chez l'animal d'expdrimentation, est bri~vement discut6e. On estime que les changements qui surviennent, traduisent l'intrins~que mode d'action de l'Atromide, qu'ils diff6rent selon l'esp~ce et qu'~ls ne portent atteinte ~t aucune fonction. ZUSAMMENFASSUNG
Der Einfluss von Atromid auf Leber- und Serumenzymaktivit~t, auf das F6tus und auf das endokrine System bei Versuchstieren, wird kurz besprochen. Die auftretenden J. A theroscler. Res., 3 (1963) 737-739
BIOCHEMICAL ASPECTS OF TOXICOLOGY OF ATROMID
Ver~nderungen werden betrachtet den
als A u s s e r u n g e n d e r i n n e r e n W i r k u n g
modifiziert
nach
unterschiedlichen
Funktionen
nicht beeintr~chtigend.
untersuchten
Arten,
und
739
des Atromid, die n o r m a l e n
REFERENCES I j. M. THORP, Lancet, i (1962) 1323. 2 W. M. FLORSHEIM, S M. VELCOFF AND A. n . WILLIAMS, Endocrinology, 72 (1963) 327.
DISCUSSION K. G. GREEN
(CHAIRMAN)
OLIVER (Edinburgh) : I would like to place on record m y opinion t h a t A t r o m i d should never be given during pregnancy, unless for a very over-riding reason. Over 8 years ago G. S. BOYD and I, and again more recently, investigated s e r u m lipid levels in p r e g n a n t w o m e n and found a considerable increase in s e r u m cholesterol, phospholipid and triglyceride in the last trimester. This is a physiological response to n o r m a l p r e g n a n c y and it would be w r o n g to t r y to alter it. GRESHAM (Cambmdge): We have heard a great deal a b o u t the results of C P I B on the lipids in h u m a n s , b u t if we are aiming at finding out the mode of action of the drug, more pertinent information m i g h t be obtained from work in animals. There are several experimental models which could be used. The r a t fed on u n s a t u r a t e d f a t t y acids develops t h r o m b o s e s which are associated with raised s e r u m triglyceride levels. W h a t would C P I B or Atromid do to these? At 0.3 % Atromid in a high fat diet for four m o n t h s , we have been able to show no effect on the d e v e l o p m e n t of lesions in the aorta, t h o u g h there was a small b u t significant drop in the s e r u m cholesterol of these animals. I do not regard r a b b i t atherosclerosis as being very similar to the h u m a n condition, however, and I suggest t h a t e x p e r i m e n t s on platelet adhesiveness in animals on high fat diets, such as the e x p e r i m e n t s described by MUSTARD, m i g h t also be relevant. THORP (I C.I.) : We have in fact looked at w h a t h a p p e n s in the r a b b i t fed on a cholesterolsupplem e n t e d diet. Simultaneous a d m i n i s t r a t i o n of A t r o m l d to such animals does reduce the cholesterol level slightly. I t is n o t a very striking thing and I would imagine in the sort of e x p e r i m e n t t h a t DR. GRESHAM is describing the effect would be even smaller. Our experience in b o t h r a b b i t s and m o n k e y s has generally been t h a t the e x t e n t of aortic atherosclerosis associated w i t h these artificially elevated s e r u m lipids is almost directly proportional to the circulating level of lipids in life W h a t we h a v e done is to take r a b b i t s and to t r e a t t h e m for a period of 12 weeks with a cholesterol s u p p l e m e n t e d diet and m a t c h t h e m at the end of this period on the basis of their s e r u m lipids. Half were r e t u r n e d to a n o r m a l ' d i e t , and half p u t on a diet s u p p l e m e n t e d with A t r o m i d for 8 weeks. The rate of regression of the s e r u m lipids and the degree of atherosclerosis were measured. There was a small b u t significant reduction in aortic a t h e r o m a and some increase in the rate of fall of the s e r u m lipids in the treated group This I t h i n k only p a r t l y answers one of the questions which one hears asked continually: where does the cholesterol go? This particular e x p e r i m e n t suggests at least t h a t it does n o t go into the aorta.
J. Atheroscler. Res., 3 (1963) 737-739