~,larine Entironmentul Research 34 { 1992j 127-131

Influence of Dietary PUFA Content on Cytochrome P450 and Transferase Activities in Atlantic Salmon

(Salmo salar)

Stephen George & James Henderson

NERC Unit of Aquatic Biochemistry, School of Natural Sciences, University of Stirling, Stifling, UK, FK9 4LA

,.t BS TR A C T

Hepatic hiotran,~/brmation actirities were meastu'ed in sahnon after maintenance fiJr 4 months on diets contahthtg different ratios ~f ( n-3 ) atut ( n- 6) polytmsatttrated./'atty acids. Qt'toehroote P4501A (etho.vyresort(fin-O- ~h'eth.vlase. EROD) activities itt fish fed h(~lt seed oil ~#ets (45?.o 18:2 (n-6 ); (n-3)/(n-6) = I).2) were thmhle and phenol UD P-glucuronosyltran.~'['erase ( L : D PG T) aet it'ities ha(['those ht [ishfed diets containing h~eh-qualit y fish oil ((n-3)/(n-6) =6). bt both groups the total cvtochrome P450 h'rels and glutathiotte-S-tran.~['erase (GST) actit'ities were constant. Analyses ~] membrane lipids showed that there were no s~gn(/icant d([]'erences in lipid chtss compositions between dietary groups: howet'et; there were marked d(ff'erences itt the Jittty acid ,vwcies composition of the phospholipids. Phosphatidyl- htositol cotttained a low (n-3)/(n-6) ratio in both groups. The fatty acid compositions of the other pho.vpholipids re/letted those of the diets: e.g. the ( n- 3)/(n-6) ratio q/'pho.v~hatidylcholhte was 1"9 in the .s'tttt[Iower oil group (compared with 21 ./or the fish oil diet). The results indicate regttlation ~f mierosomal hiotran,sJ'orntation aetit'ities h v tfietary modification of the fatty acid composition ~71" the enth~plasmic retieuhtm htt[]< phospholipids.

The activities of integral membrane enzymes such as the hepatic microsomal drug and carcinogen-metabolising systems are regulated by the intact phospholipid structt, re of the membrane L'z and can be modified by dietary- induced changes in membrane fluidity, phospholipid/cholesterol ratios and [.ttty acid compositions. 3''~ Most hepatic microsomal enzyme systems, including the mixed function oxygenases (MFOs), transferases and phosphatases require associated enzymes, electron transport proteins or

127

Marine Enriron. Res. 0141- I 136/92/$05.00 t~ 1992 Elsevier Science Publishers Ltd, England. Printed in Great Britain

128 Stephen George. James Henderson

translocases and phospholipid for functionality. Thus, in such complex multi-component systems the mechanistic basis for membrane-dependent regulation of activities is largely unknown and is also complicated by recent findings that the expression of MFOs, for example, is transcriptionally regulated by many dietary components, including vitamins and natural and synthetic antioxidants, s In this preliminary study we report the effects of dietary manipulation of the fatty acid composition of the microsomal phospholipids on components of the hepatic biotransformation system of salmon liver.

Groups of 150 Atlantic sahnon {average initial wt 15"5 g) were maintained in freshwater at 8: C for 4 months and fed experimental diets containing: fish meal, 70g: carboxymethylcellulose, 10g: dextrin, 4g: vitamin mix, 6 2g: mineral mix, 6 4g: and antioxidant mix. 6 0"8 g. with either 10g fish oil (Seven Seas Ltd, Hull, UK)((,-3) diet, 15.4% lipid) or 10 g sunflower oil (Seven Seas Ltd) ((,-6) diet, 14.5% lipid) per 100g dry diet. Fish were sacrificed, livers removed and microsomes prepared as described previously. 7 Aliquots were analysed for lipid class and fatty acid compositions, 8 protein, cytochrome P450 and enzyme activities. Q Cytosols were assayed for protein and GST. 7

After 4 months dietary treatment, livers from groups of three fish [i.e. l\mr samples per treatment group) were pooled and fractionated. The microsomal total lipid contents and their class compositions were the same ['or both dietary groups, the phospholipid/cholesterol molar ratios were approximately 2 and phospholipids comprised the major component {57%), of which 30% was phosphatidylcholine (PC). The fatty acid (FA) composition of phosphatidylinositol (PI) was the same for both dietary groups: 18:0, 20:4(,-6) and 22:6 (n-3) comprising 78% of the FAs and {,-3)/ {,-6) polyunsaturated fatty acid (PUFA) ratios of 0"8 (data not shownl. The FA composition of the other phospholipids was highly dependent upon the diet, as shown for the major component, PC, in Table 1. The total PUFA contents were not significantly different but the species compositions differed markedly. The major PUFAs for the group on the fish oil diet were 20:5(,-319% and 22: 6(n-3)40%, with a ratio of(n-3)~(,-6) of 21. While there was still 4% 20:5(,-3) and 31% 22:6(,-3) in the PC of the sunflower oil led group there was a large increase in 18:2(n-6) to 14% (compare with <1%). Thus the (,-3)/(n-6) ratio was only 2.

There were no significant differences in size, body or liver weights between the two groups offish (Table 2). The microsomal and cytosolic proteins, total cytochrome P450 contents and cytosolic GST activities were not significantly different. However, there were marked differences in micro- somal biotransformation activities in fish fed different diets (Table 2). EROD activities were significantly higher ( 1.7-fold, P < 0-05) in the group fed the (n- 6)-rich diet compared with those fed the (n-3)-rich diet. While associated reductase activities (NADH b5- and NADPH cytochrome c-reductase) were

Influence o f dietary PUFA content in .4 t/untic salmon 129

TABLE i Fatty Acid Composition (wt%) of Experimental Diets and Microsomal Phosphatidylcholine IPCJ in Atlantic Salmon after 4 Months Treatment. Values for Individual Selected Major

Components and Summarised Class Data ~

Fatty acid [selected)

Fish oil, (n-3)-rich diet Sunflower oil, (n-6)-rich diet

Diet Microsomal PC Diet Microsomal PC

16:0 190_+0.5 28.4_,+2.3 10.8-+0'2 26"8-+2"7 16:Iln-71 8'4 _+ 0'2 3-9 __ 19 2"4 +0"1 0'9 -+0"8 I ~: 11/1-91 10-7+0.4 8.2_+0"7 19-3_+0"3 91 _+0.4 I ~:21n-61 1"9 4- 0" I 0"7 +_ 0" I 39'7 -+ 0"7 3"8 +_ 0"6** 22:61n-3) 9"1 + I'1 39"7 + 2"6 3"~ -+0"3 31"3 -+ 2'0**

f~tt v acid classes Saturates 30-9 31"8 + 18 18-2 29. I + 1"6 Monounsaturatcs 35-8 14-6 + 0'9 31-9 14.5 + 0.6 In-71 12 3.9 + I-9 2-6 2.0 + 0-4 In-9) I 1.7 9.8 +0-5 19-7 10.3 +0.3 (n-I II 6-0 0.1 __+ 0-1 56 0-2-+0-1 PUFA 330 53"3 _+ 2"9 49"9 56"4 _+ 1"7 (H-3) 257 50"9 + 3"0 8'5 36'6 _+ 2"8** In-6) 4"3 2'4 -+ 0'2 40"6 19'8 -+ 1"4"*

(n-3),Or-6) 60 20-9 + 27 02 1"9 + 03**

J T~velvc lish per treatment group maintained on experimental diets for 4 months; livers from groups of three animals pooled. Means _ SD, on three samples of diets or four liver samples; significant differences in microsomal PC composition established by two-tailed Student's t- test at P < 0"05**.

some 1.4-fold higher, the differences were not statistically significant. In contrast, phenol UDPGT activity, measured in native non-disrupted microsomes with l-naphthol as acceptor substrate, was 2-fold higher in the group fed the (n-3)-rich diet.

Most previous studies, both in mammals and in fish, have not been carried out with very highly purified dietary lipid components and controlled levels of antioxidants and, in view of the high susceptibility of these poly- unsaturate-rich oils to peroxidation and of the inductive effects of antioxidants, comparisons are difficult. The present results contrast with those reported for rats, in which animals led {n-3)-rich fish oils displayed 20- 30% higher MFO activities towards several substrates, including benzo(a)- pyrene, t°'lt and for channel catfish. ~2 In the catfish study it was reported that cytosolic GST activity and cytochrome P4501A-dependent activities (EROD and ECOD) were higher after feeding rations containing (n-3)-rich fish oils compared with (n-6)-rich corn oils; however, inter-animal variations were greater than mean differences--liver somatic indices and lipid contents

130 Stephen George, James Henderson

TABLE 2 Effect of Dietary PUFA Composition on Hepatic Biotransformation Systems of Atlantic

Salmon"

Parameter Fish oil diet Sut~ower oil diet

Length. cm 13 + 1.4 13 +0.8 Body wt, g 26 + 9 24 + 46 Liver wt, nag 320 + 120 290 + 60 LSI, h % body v,t 1.2 _ 0-28 13 4- 0.3 Microsomal protein, mg/g 32.5 _+ 10.1 24.8 4-4.4 Cytosol protein, mg, g 52 + 16 55 4- 8 Total P450, nmol, mg 0.34 4- 0-09 0.38 _+ 0.06

NADH b5-reductase5/tmol"min, mg NADPH cyt. c-reductasc, nmol,min'mg EROD, nmo!.min, mg Phenol UDPGT, a nmol rain nag GST. ,umol, rain nag

045 + 004 063 ___ 0-15 7"5 + 2.4 10-6 + 1'3

0"54 + 0'15 1"33 _____0.17"* 2"4 __+ 0"8"* 1-45 + 0-3

0"56__+0-19 0-4____.0"06

Twelve fish per treatment group ~verc maintained on experimental diets ff, r 4 months; li~ ors from groups of three lish were pooled for analysis. LSI, li,,cr v,t as %, of bod~ wt.

' Measured by reduction of fcrric vanidc. J ,\leasurcd with I-naphthol as acceptor substratc. Values. means +_ SI) I\~r four samples of pooled microsomcs from three indi',idtlals each; ,;ignilicant difl'crcnccs established by t~,o-tailed Student's t-test at t '< 0"05**.

were different. Thus, in terpreta t ion and compar i son is difficult, especially

since G S T activity is not l ipid-dependent and is induced by dietary

ant ioxidants such as BHA, e thoxyqu in and BHT. 7

F rom the present s tudy it canno t be determined whether differences in the

activities measured in ritro are direct effects o f enzyme expression or o f the

m e m b r a n e lipid envi ronment . Both purified c y t o c h r o m e P450s and

U D P G T s require added lipid (PC) for reconst i tut ion o f maximal activity;

thus, the observed effect may be direct or result from alterat ions in substrate

diffusion. Both enzyme systems also involve addit ional membrane proteins

for activity in microsomes and changes in expression or activity o f any of

these c o m p o n e n t s could alter the apparen t activity o f the systems. Fur ther

studies are therefore required to address such questions.

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Influence of dietary PUE4 content in ,-1 tluntic salmon 131

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