Long-term toxicity studies on chocolate brown HT in rats

Toxicology, 11 (1978) 303--307 O Elsevier/North-Holland Scientific Publishers Ltd.

L O N G - T E R M TOXICITY S T U D I E S ON C H O C O L A T E BROWN HT IN RATS FRANCIS M.B. CARPANINI, KENNETH R. BUTTERWORTH, IAN F. GAUNT, IDA S. KISS, PAUL GRASSO and SHARAT D. GANGOLLI

The British Industrial Biological Research Association, Woodmansterne Road, Carshalton, Surrey SM5 4DS (Great Britian) (Received June 8th, 1977) (Accepted September 13th, 1978) SUMMARY

Groups o f 48 male and 48 female rats were given diets containing 0 (control), 500, 2000 or 10 000 ppm Chocolate Brown HT for 2 years. These treatments had no adverse effect on mortality, body-weight gain, food or water consumption, haematology, renal function, serum constituents, organ weight or histopathology. From the incidence of tumours observed in the control and test animals it is concluded that Chocolate Brown HT did n o t exert any carcinogenic effect and that the no-untoward-effect level was 10 000 ppm.

INTRODUCTION

Chocolate Brown HT (C.I. (1971) No. 20285) is the disodium salt o f 4,4'[ (2,4-dihydroxy-5-(hydroxymethyl)-m-phenylene)bis(azo)] di-l-naphthalenesulphonic acid. It is a water-soluble colouring, which is permitted for use in food in the UK under the Colouring Matter in F o o d Regulations [1] and its review by the EEC authorities is pending. The legislative position and metabolic and toxicological data pertaining to Chocolate Brown HT have been summarized by Drake, Butterworth, Gaunt and Hardy [ 2 ] . Drake et al. [2] carried o u t a long-term study in mice with dietary levels of 0.01--0.5% Chocolate Brown HT and established a no-untoward-effect level above 140 mg/kg/day. No evidence o f carcinogenic potential was found at levels up to 700 mg/kg/day, and as certain debatable findings in mice given the highest level of t r e a t m e n t were probably unrelated to the administration of the colouring, it is possible that the true no-untoward-effect level This is an abridged paper. Copies of the full paper are available from the Editor on request, which should be accompanied by $5.00, or equivalent, to cover reproduction and postage.

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was considerably in excess of the stated figure. Hendy, Butterworth, Gaunt, Hooson and Grasso [3] fed the colouring in doses of 5--100 m g / k g / d a y for 13 weeks to pigs w i t h o u t adverse effects. They obtained a no-untowardeffect level of at least 100 mg/kg/day. The present report describes a long-term toxicity and carcinogenicity study in rats. It completes the investigations on Chocolate Brown HT carried o u t as part of BIBRA's safety evaluation programme. MATER I ALS AND METHODS

Materials Chocolate Brown HT was supplied through the Food Colours Committee of the Chemical Industries Association and complied with the specification of the Joint FAO/WHO Expert Committee on Food Additives [ 4 ] . It had the same specifications as the material used by Drake et al. [ 2 ] . Animals and diet Wistar-derived rats, obtained from a specified pathogen-free breeding colony, were given ground Spratts Laboratory Diet No. 1 and water ad lib. They were housed, 4 per cage, in a room maintained at 20 + 1°C with a , relative h u m i d i t y of 50--60%. Experimental design and conduct Chocolate Brown HT was incorporated at 0 (control) 500, 2000 or 10 000 ppm in the diet of groups of 48 male rats (body wt. 54--80 g) and 48 females (body wt. 53--82 g) for 2 years. Individual body weights were recorded on the first day of feeding, at week 1, 4, 7 and 11 of t r e a t m e n t and thereafter at approx. 3-monthly intervals up to week 102. Food intake was measured over the 24-h period preceding each weighing and the water consumption was measured over the 48-h period commencing at the same time as the food intake. At week 14, 27, 55 and 80 o f the study, blood was collected from the tail veins of 10 male and 10 female rats from each of the groups fed diets containing 0, 2000 or 10 000 ppm Chocolate Brown HT. All the samples were examined for haemoglobin content, packed cell volume and counts of erythrocytes and total leucocytes. Reticulocyte and differential white cell counts were carried out in blood samples from control rats and those given 10 000 ppm Chocolate Brown HT in the diet for 14 and 27 weeks only. Samples of blood obtained from all the animals at the end o f the experiment were subjected to a similar examination, again excluding counts of reticulocytes and individual types of leucocytes. At weeks 14, 27 and 56, a urinary concentration test was c o n d u c t e d on 10 male and 10 female rats from each o f the groups given diets containing 0 or 10 000 ppm Chocolate Brown HT. At week 102 the test was c o n d u c t e d on 10 male and 10 female rats from each of the t r e a t m e n t levels and the controls. In all of these studies, measurements were made of the specific

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gravity and volume of urine produced during a 6-h period of water deprivation, over a 4-h period commencing after 16 h w i t h o u t water and over a 2-h period following a water load o f 25 ml/kg b o d y wt. At the same time samples of the urine were examined for appearance, microscopic constituents and c o n t e n t o f cells, glucose, ketones, bile salts and blood. After week 104 all the surviving animals were killed by exsanguination under barbiturate anaesthesia following an overnight period w i t h o u t food. The blood obtained was used for the terminal haematological studies and serum chemistry. Serum was analysed for its c o n t e n t of urea, glucose, total protein and albumin and for the activities of glutamic-oxalacetic transaminase, glutamic-pyruvic transaminase and lactic dehydrogenase. At autopsy any macroscopic abnormalities were noted and the brain, heart, liver, spleen, kidneys, stomach, small intestine, caecum, adrenals, gonads, pituitary and thyroid were weighed. Samples of these organs, together with samples o f all major tissues and organs and of any other tissue appearing abnormal at autopsy were preserved in 10% buffered formalin until prepared for histological examination. Statistical calculations are based on a level of significance of at least P = 0.05. RESULTS Cumulative mortality was similar in the treated and control animals t h r o u g h o u t the 2-year period except for one statistically significant value at week 72 (7 deaths compared with none in the controls) in males given 10 000 ppm Chocolate Brown HT. The only statistically significant differences in body weight were seen in the rats given the 500 ppm level of treatment. These values were lower than those for the corresponding controls and were recorded at week 102 in males and week 78 in females. There were no statistically significant differences between treated and control animals in food or water intake. The approximate total intakes of colouring consumed per rat up to week 66 were for the low, medium and high t r e a t m e n t levels respectively, 5.25, 21.5 and 109.8 g by the males and 4.15, 17.0 and 83.9 g by the female rats. The corresponding values up to week 102 were 8.11, 32.3 and 160.8 g/rat by the males and 6.38, 21.1 and 128.2 g/rat for the females. Total intakes by week 66 were calculated separately because 97% of the rats were still alive at this time. There were several isolated statistically significant differences between treated and control rats in the results of the interim haematological examinations. These consisted of a reduced haemoglobin concentration at week 55 in males and females on the highest level of treatment, an increased red cell c o u n t in males given 2000 ppm Chocolate Brown HT for 14 and 27 weeks, in males given 10 000 ppm for 27 weeks and in females given 2000 ppm for 55 weeks, and a reduced red cell c o u n t in females fed diets containing 2000 or 10 000 ppm for 27 weeks. In addition, the white cell counts were higher than the control value in females given 10 000 ppm of the colouring for 14 weeks and lower in males given 2000 ppm for 27 weeks and females given

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2000 ppm for 55 weeks. However, there were no statistically significant differences between treated and control animals in the terminal haematological studies. The results of the urinary studies and serum analyses showed no significant differences between treated and control animals. Apart from a slight reduction in testis weight in rats given 500 ppm Chocolate Brown HT in the diet, there were no statistically significant differences between the absolute organ weights of the treated and control rats. The only statistically significant differences between treated and control rats in relative organ weights were slightly higher relative spleen and kidney weights in males given diet containing 10 000 ppm Chocolate Brown HT and a slightly higher relative spleen weight in females given 500 ppm. There was a wide range of pathological changes, particularly in the liver and kidney although, with the exception of a lower incidence of f a t t y change in the livers of males given 2000 or 10 000 ppm of the colouring compared with the controls, the incidence of these lesions was similar in both test and control groups. There was also a high incidence of adenosis and fibroadenosis in the female rats, but the incidences in the treated and control animals showed no statistical differences. The incidence of tumours was low in all groups, the m o s t c o m m o n tumours being m a m m a r y adenomas and fibroadenomas in the female rats, interstitial cell tumours of the testis, and subcutaneous fibromas in both sexes. The incidence of these findings in the treated and control animals did n o t differ statistically. Very few of the tumours were malignant. On examination of all tissues both at autopsy and in the course of the microscopic study there was no indication of pigmentation or storage phenomena. DISCUSSION

Although the n u m b e r of deaths among the male rats given the highest level of t r e a t m e n t for 72 weeks was significantly higher than t h a t in the controls at this time, there were no statistically significant differences between treated and control animals t h r o u g h o u t the rest of the study and the overall mortality rate was n o t affected by t r e a t m e n t with Chocolate Brown HT. The scattered changes detected in the haematological studies at weeks 14, 27 and 55 were n o t related to t r e a t m e n t and none of these differences were evident at weeks 80 or 104. No effects attributable to t r e a t m e n t were detected by urine examination, renal concentration tests or serum analysis. In rats showing some reduction in testis weight (those on the 500 ppm treatment) no lesions differing in type or incidence from those in control rats were found on histological examination. Moreover, the incidence of testicular tumours was lower in this group than in the control rats. Thus this decreased testicular weight c a n n o t have been related to treatment. The slightly increased relative spleen and kidney weights seen at the top treatm e n t level in males were n o t associated with any pathological findings, nor were these increases evident in females except in the spleens of those on the

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lowest t r e a t m e n t level. These differences are unlikely, therefore, to have been related to treatment. The histopathological changes were those expected in ageing rats, and apart from the lower incidence o f f a t t y change seen in the livers o f rats given the 2 higher dietary levels of the colouring, their incidence and severity were similar in both test and control rats. The incidence o f t u m o u r s in this s t u d y was low and was clearly unrelated to treatment, being similar in both test and control groups. The absence o f discolouration of the intestines and l y m p h nodes at p o s t m o r t e m examination contrasted with observations in mice exposed to Chocolate Brown HT [2]. The present observations do n o t allow any explanation o f this difference, although metabolism of the colouring by intestinal bacteria is likely to be different in the 2 species. No long-term toxicity or carcinogenic potential has been detected with dietary levels of Chocolate Brown HT up to 10 000 ppm. This no-untowardeffect level is equivalent to an intake of approximately 500 m g / k g / d a y given over most of the lifespan of the rats. REFERENCES 1 Colouring Matter in F o o d Regulations 1973. S t a t u t o r y Instrument 1973, no. 1340. 2 J.J-P. Drake, K.R. Butterworth, I.F. Gaunt and J. Hardy, Toxicology, 10 (1978) 3--12. 3 R.J. Hendy, K.R. Butterworth, I.F. Gaunt, J. Hooson and P. Grasso, Toxicology, in press. 4 Joint F A O / W H O Expert C o m m i t t e e on F o o d Additives, Eighth Report, Tech. Rep. Set. Wld. Hlth. Org., 309 (1965).

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ing number of investigations since the disastrous incident in Michigan, where livestock and poultry on hundreds of farms became exposed to PBBs [1]. PBBs, used as fire retardants, resemble in many ways the structurally related PCBs, e.g., PBBs cause liver enlargement in mammals [2--4], they enhance drug elimination from plasma [4], and have been reported to be weakly teratogenic [2]. High concentrations of orally administered PBBs are excreted in milk by ruminants [ 5] ; both PCBs and PBBs are found in milk of lactating humans [6]. PCBs are known to induce the activities of enzymes catalyzing drug biotransformation [ 7 - 1 1 ] . Itecent studies have shown that PBBs enhance the activities of hepatic enzymes involved in drug hydroxylation [12--15] and epoxide hydration [12,14]. However, it is not known what effects the PBBs have on enzymes catalyzing the second phase of drug biotransformation, the conjugation reactions. Moreover, there is little information about the effects of PBBs on tissues other than liver. This study was carried out with 2 industrial PBB-mixtures to find answers for these questions: (i) do PBBs share the properties of PCBs even with respect to glucuronidation and glutathione conjugation; (ii) what are the effects of PBBs o n drug metabolizing enzymes in the lung and in the kidney. MATERIALS AND METHODS The 2 industrial bromobiphenyl-mixtures, Firemaster BP-6 ("hexabromobiphenyl", Michigan Chemical) and Fit 250 13A ("octabromobiphenyl", Dow Chemical) were purchased from EnChem Environmental Division, ItFit Corporation, Hope, R.I., USA. Firemaster BP-6 consists principally of hexa(67%) and heptabromobiphenyls (25%) [16]. The most abundant isomer is 2,4,5,2',4',5'-HBB [17]. The main components of Fit 250 13A are nona(54%) and OBBs (38%) [16]. A mixture of PCBs (Clophen A 50) was obtained from Bayer A.G., Leverkusen, GFIt. Adult male C57 mice weighing about 30 g were used. PBBs (75 mg/kg) dissolved in corn oil were injected intraperitoneally to mice (5 ml/kg) as a single dose. Control animals received an equal volume of corn oil. Enzyme activities were assayed 10 days after the injection of PBBs. The microsomes were isolated by Ca2+-aggregation [18,19]. The treatment of microsomes with digitonin was carried out as described by H~inninen [20]. The activity of AHH (EC 1.14.14.2) was measured with the radiometric method of DePierre et al. [21]. Ethoxycoumarin deethylase was assayed with the fluorimetric method of Ullrich and Weber [22] as modified by Aitio [23]. Glutathione S-transferase activity was measured in the postmicrosomal supernatant as described by James et al. [24]. Epoxide hydratase (EC 4.2.1.63) activity was determined with the method of Oesch et al. [25] with [3H]styrene oxide as the substrate. The activity of UDP-glucuronosyltransferase (EC 2.4.1.17.) was measured with 4-methylumbeUiferone as the

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aglycone [26,27]. Protein content was measured with the biuret method

[28]. The statistical analysis of results was carried out using Student's t-test. RESULTS

HBB and OBB increased the activity of hepatic AHH 1.9- and 1.5-fold, respectively. Ethoxycoumarin deethylase activity in the liver was enhanced by HBB (5.7-fold) and by OBB (2.4-fold), whereas ethoxycoumarin deethylase activities in other tissues of bromobiphenyl-treated animals did not differ from those of control animals (Table I). TABLE I THE E F F E C T OF A SINGLE I N T R A P E R I T O N E A L INJECTION OF POLYBROMIN A T E D BIPHENYLS ON HEPATIC A R Y L H Y D R O C A R B O N H Y D R O X Y L A S E AND E T H O X Y C O U M A R I N D E E T H Y L A S E A C T I V I T Y IN D I F F E R E N T T I S S U E S O F C57 MICE a Enzyme Aryl h y d r o c a r b o n hydroxylaseb Ethoxycoumarin Deethylaseb

a b c d e

Tissue

Liver Liver Kidney Lung

Control

Hexabromobiphenyl

4.10 -+ 0.50 41.2 -+ 4.6 2.18 -+ 0.31 0.41 +- 0.12

7.71 -+ 0.97 d 234.3 -+ 35.2 e 2.49 +- 0.36 0.60 ± 0.20

Octabromobiphenyl

6.00 -+ 0.65 c 97.1 -+ 9.9 e 1.88 -+ 0.18 0.29 -+ 0.05

M e a n results -+ s t a n d a r d e r r o r o f m e a n s are i n d i c a t e d , 8 a n i m a l s / g r o u p . E n z y m e activity is given as n m o l / m i n X g w e t wt. 2P < 0.05. 2P < 0.01. 2P < 0.001.

T A B L E II LIVER, KIDNEY AND LUNG GLUTATHIONE S-TRANSFERASE, AND LIVER E P O X I D E H Y D R A T A S E A C T I V I T I E S IN C57 MICE A F T E R I.P. A D M I N I S T R A T I O N OF POLYBROMINATED BIPHENYLS a Enzyme Glutathione S-Transferaseb

Epoxide hydratase b a b c d e

Tissue

Control

Liver Kidney Lung Liver

11000 3170 1200 71.7

Hexabromobiphenyl Octabromobiphenyl

-+ 432 -+ 225 -+ 193 _+ 9.8

18300 2840 1000 110.0

+- 863 e -+ 277 -+ 159 -+ 6.2 d

15300 2360 1360 67.7

± 1030 d -+ 197 c +- 268 -+ 3.4

Mean results -+ s t a n d a r d e r r o r o f m e a n s are i n d i c a t e d , 8 a n i m a l s / g r o u p . E n z y m e activity is given as n m o l / m i n × g w e t wt. 2P < 0.05. 2P < 0.01. 2P < 0.001.

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T A B L E II I THE E F F E C T OF B R O M O B I P H E N Y L S ON U D P - G L U C U R O N O S Y L T R A N S F E R A S E A C T I V I T Y a IN D I F F E R E N T T I S S U E S O F C 5 7 M I C E b Tissue (treatment of microsomes) Liver Non-activated Digitonin-activated Kidney Non-activated Lung Non-activated

Control

86.4 + 5.8 374 + 16

Hexabromobiphenyl

Octabromobiphenyl

1 2 9 .7 + 12.7 d 544 + 24 e

9 2 . 5 -+ 10.9 450 -+ 22 c

19.8 ±

2.3

29.9 ±

1.8 d

27.7 ±

3.7

20.4 ±

2.5

20.0 ±

1.3

21.4 ±

1.2

a U D P - G l u c u r o n o s y l t r a n s f e r a s e a c t i v i t y is d e f i n e d as n m o l 4 - m e t h y l u m b e l l i f e r y l g l u c u r o n i d e f o r m e d / r a i n X g w e t wt. E n z y m e a c t i v i t y in t h e liver was m e a s u r e d f r o m n a t i v e m i c r o s o m e s an d f r o m m i c r o s o m e s a c t i v a t e d b y d i g i t o n i n t r e a t m e n t . K i d n e y a n d lung UDP-glucuronosyltransferase activities were measured from non-activated microsomes. b Me an s -+ S E M f r o m 8 a n i m a l s p e r g r o u p . c 2P < 0.05. d 2P < 0.01. e 2P < 0.001.

In the liver both HBB and OBB increased the glutathione S-transferase activity (1.7- and 1.4-fold, respectively); in the kidney OBB caused a slight but statistically significant decrease in glutathione S-transferase activity; in the lung brominated biphenyls had no effect on the activity of glutathione S-transferase. Hepatic epoxide hydratase was enhanced by HBB (1.5-fold) but not by OBB (Table II). HBB treatment caused a 1.5-fold increase in the activity of UDP-glucuronosyltransferase when measured from either native or digitonin-treated liver microsomes (Table III). A slight induction of hepatic UDP-glucuronosyltransferase was seen in OBB-treated mice when the enzyme activity was measured from digitonin-treated microsomes. UDP-glucuronosyltransferase activity increased 1.5-fold in native microsomes of the kidney from HBBtreated mice; bromobiphenyls had no effect on pulmonary UDP-glucuronosyltransferase (native microsomes). Similar enhancement of enzyme activity was seen in the liver of mice pretreated with a mixture of PCBs (Clophen A 50): AHH activity increased 2.5-fold (n = 3), ethoxycoumarin deethylase 5.4-fold (n = 3), glutathione S-transferase 1.7-fold (n = 3), epoxide hydratase 1.7-fold (n = 3), UDPglucuronosyltransferase from native microsomes 1.4-fold (n = 3) and from digitonin-treated microsomes 1.5-fold (n = 3). DISCUSSION

This study indicates that Firemaster BP-6, a mixture of PBBs consisting

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mainly of HBB is an inducer of drug metabolizing enzymes. Dent and coworkers have shown that Firemaster BP-6, given either intraperitoneally [12] or as a dietary supplement [ 1 3 , 1 4 ] , enhances the activities of enzymes catalyzing drug h y d r o x y l a t i o n and epoxide hydration in rat liver. A pure HBB isomer, 3,4,5,3',4',5'-HBB when injected intraperitoneaUy to rats or mice is reported to cause an increase in A H H activity [ 1 5 ] . The data presented in this paper reveal a further analogy in the nature of PBBs and PCBs: Firemaster BP-6 also increased the activities of glutathione S-transferase and UDP-glucuronosyltransferase, enzymes catalyzing conjugation reactions in drug biotransformation. We f o u n d that " F R 250 13A", a mixture which is sold as " o c t a b r o m o b i phenyl", is also an inducer of drug metabolizing enzymes. The e x t e n t of enzyme induction caused by F R 250 13A was, however, generally lower than that caused b y Firemaster BP-6. This suggests that OBBs are less p o t e n t inducers of drug metabolizing enzymes than HBBs. Final conclusions on the p o t e n c y of different isomers, however, await studies p e r f o r m e d with pure b r o m o b i p h e n y l isomers. Brominated biphenyls had little effect on drug metabolizing enzymes in tissues other than liver. In the kidney Firemaster BP-6 increased UDPglucuronosyltransferase activity to the same e x t e n t as in the liver. Dent et al. [14] have f o u n d that Firemaster BP-6, when given to rats in the diet, enhances m a m m a r y activity o f A H H b u t decreases that of epoxide hydratase. A PCB mixture (Clophen A 50) has been reported to enhance the activity of A H H in the kidney and in the lung of the rat [ 1 0 ] . C57 is a so called responsive mouse strain, i.e., the hepatic A H H activity is enhanced after administration of polycyclic aromatic h y d r o c a r b o n s [ 2 9 ] . In the present s t u d y it was shown that the C57 mice also respond with induction of drug metabolizing enzymes to t r e a t m e n t with PBBs. F r o m the results of this investigation it can be concluded that the effects of PBBs on drug metabolizing enzymes are comparable to the effects described for PCBs. In this respect it seems to be of minor importance whether the biphenyl is substituted with chlorine or with bromine.

ACKNOWLEDGEMENTS

The technical assistance of Ms. Raija S6derholm and Ms. Leena R u o k o n e n is gratefully acknowledged. This research was financially supported by grants from the Juho Vainio F o u n d a t i o n , Finland and N.I.H. R O I - E S O 1 6 8 4 (U.S.A.).

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3 K.P. Lee, R.R. Herbert, H. Sherman, J.G. Aftosmis and R.S. Waritz, Toxicol. Appl. Pharmacol., 34 (1975) 115. 4 S.Z. Cagen, M.M. Preache and J.E. Gibson, Toxicol. Appl. Pharmacol., 40 (1977) 317. 5 W.H. Gutenmann and D.J. Lisk, J. Agric. Food Chem., 23 (1975) 1005. 6 Final Report of the Subcommittee on the Health Effects of Polybrominated Biphenyls and Polychlorinated Biphenyls, July, 1976, Department of Health, Education and Welfare, Washington, D.C. 7 A.H. Conney, Pharmacol. Rev., 19 (1967) 317. 8 D.J. Ecobichon and A.M. Comeau, Chem.-Biol. Interact.,9 (1974) 341. 9 H. Vainio, Chem.-Biol. Interact.,2 (1974) 7. 10 H. Vainio, Chem.-Biol. Interact.,2 (1974) 379. 11 M.G. Parkki, J. Marniemi and H. Vainio, J. Toxicol. Environ. Health, 3 (1977) 903. 12 J.G. Dent, K.J. Netter and J.E. Gibson, Toxicol. Appl. Pharmacol., 38 (1976) 237. 13 J.G. Dent, K.J. Netter and J.E. Gibson, Res. C o m m u n . Chem. Pathol. Pharmacol., 13 (1976) 75. 14 J.G. Dent, U. Roes, K.J. Netter and J.E. Gibson, J. Toxicol. Environ. Health, 3 (1977) 651. 15 A. Poland and E. Glover, Mol. Pharmacol., 13 (1977) 924. 16 J.J. DeKok, A. DeKok, U.A.Th. Brinkman and R.M. Kok, J. Chromatogr., 142 (1977) 367. 17 L.W. Jacobs, S-F. Chou and J.M. Tiedje, J. Agric. F o o d Chem., 24 (1976) 1198. 18 S.A. Kamath and K.A. Narayan, Anal. Biochem., 48 (1972) 59. 19 A. Aitio and H. Vainio, Acta Pharmacol. Toxicol., 39 (1976) 555. 20 O. H~nninen, Ann. Acad. Sci. Fenn. Ser A II, 142 (1968) 1. 21 J.W. DePierre, M.S. Moron, K.A.M. Johannesen and L. Ernster, Anal. Biochem., 63 (1975) 470. 22 V. Ullrich and P. Weber, Hoppe-Seyler's Z. Physiol. Chem., 353 (1972) 1171. 23 A. Aitio, Anal. Biochem., 85 (1978) 488. 24 M.O. James, J.R. Fours and J.R. Bend, Biochem. Pharmacol., 25 (1976) 187. 25 F. Oesch, D.M. Jerina and J. Daly, Biochim. Biophys. Acta, 227 (1971) 685. 26 I.M. Arias, J. Clin. Invest., 41 (1962) 2233. 27 A. Aitio, Int. J. Biochem., 5 (1974) 325. 28 E. Layne, Spectrophotometric and turbidimetric methods for measuring proteins, in S.P. Colowick and N.O. Kaplan (Eds.), Methods in Enzymology, Vol. 3, Academic Press, New York, 1957, p. 447. 29 D.W. Nebert, N. Considine and I.S. Owens, Arch. Biochem. Biophys., 157 (1973) 148.

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