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A study of the hepatic microsomal epoxide hydrolase in sea birds
Comp. Biochem. Physiol. Vol. 73C, pp. 463 467, 1982 Printed in Great Britain
0306-4492/82/060463-05503.00/0 © 1982 Pergamon Press Ltd
A S T U D Y O F THE H E P A T I C M I C R O S O M A L H Y D R O L A S E IN SEA BIRDS
EPOXIDE
G. C. KNIGHT and C. H. WALKER Department of Physiology and Biochemistry, The University, Whiteknights, Reading, Berks. RG6 2A J, UK (Received 3 March 1982) Abstract--l. Epoxide hydrolase activities were measured in six species of sea birds using the chlorinated epoxide HEOM as substrate. 2. The activities found fell within the general range for birds, and there were no clear sex or species differences. 3. Relatively low activities were found in liver microsomes of female puffins early in the breeding season, but high activities were found in 11,000 g precipitates from these individuals. Apart from this no correlations were found between activity and time of year, PCB residues or geographical location. 4. These results are discussed in relation to mono-oxygenase activities in the same samples.
INTRODUCTION Hepatic microsomal epoxide hydrolase can convert a wide range of exogenous and endogenous epoxides into trans diols. Substrates of the enzyme include the epoxides of polycyclic aromatic h y d r o c a r b o n s a n d steroids (Oesch, 1973; Bindel et al., 1979), insect juvenile h o r m o n e s and analogues thereof (see Wilkinson, 1976), and chlorinated insecticides such as dieldrin (Brooks et al., 1970). An i m p o r t a n t role of the enzyme is the hydration of epoxides generated in the endoplasmic reticulum by the action of cytochrome P4s0-dependent mono-oxygenase upon unsaturated substrates. In this way the cell may be protected against the toxic action of certain active oxides (e.g. of polycyclic a r o m a t i c hydrocarbons) which are formed in vivo. The enzyme is clearly of interest in connection with pollution problems. Apart from the question of the deactivation of potentially harmful epoxides referred to above, epoxide hydrolase attack can provide an effective mechanism for elimination of certain substances which are liable to undergo bioaccumulation. Thus, trans diols formed from dieldrin or its analogues, or from epoxides of polychlorinated biphenyls generated in situ, are readily excreted, usually as conjugates. The present investigation was concerned with the activity of this enzyme in fish-eating sea birds, a group with a m a r k e d tendency to bioaccumulate liposoluble pollutants. The status of hepatic microsomal mono-oxygenase was studied in the same specimens, and these results are reported elsewhere (Knight & Walker, 1982). MATERIALS AND METHODS Animals All the birds used in this study were adults. The sea birds were of the following species: the puffin (Fratercula arctica), razorbill (Alca torda) and guillemot (Uria aalge) of * HEOM = 1,2,3,4,9,9-hexachloro-l,4,4a,5,6,7,8,8a-octahydro-6,7,-epoxy- 1A-methanonaphthalene. 463
the family Alcidae, order Charadriformes; the shag (Phalacrocorax aristotelis) and cormorant (Phalacrocorax carbo) of the family Phalacrocoracidae, order Pelecaniformes; and the Manx shearwater (Puffinus puffinus) of the family Procellariidae, order Procellariiformes. Collections were made, under licence, at approximately one month intervals during the period May-July of 1978-1980 from each of the following sites: The Saltee Islands, Co. Wexford, Eire (52 ° 7'N. 6 ° 35'W) and the Isle of May, Fife, Scotland (56 ° l l'N, 2 ° 33'W). A single collection was also made from St. Kilda, Outer Hebrides, Scotland (57 ° 50'N, 8 ° 40'W) in June 1979. The puffin and Manx shearwater were the only species taken from St. Kilda; all cormorants came from the Saltee Islands. After capture in the breeding colonies, the birds were transported back to the laboratory in large, well-ventilated plastic containers, and maintained on a diet of sprats (Sprattus sprattus), with an adequate supply of water, until used for experimentation (usually within 5 days of capture). Cormorants were frequently maintained for longer periods. Adult male Sprague-Dawley rats (300400g) were maintained as previously described (Chipman & Walker, 1979). Rooks (Corvus frugilegus) and black headed gulls (Larus ridibundus) were obtained from the Pest Infestation Control Laboratory, Ministry of Agriculture, Fisheries and Food, Tolworth, Surrey and had been caught in the wild. Substrate and metabolite HEOM* and its metabolite HEOM trans-diol were prepared as previously described (Craven et al., 1976; Craven, 1977). HEOM in ethanolic solution (2 mg ml-1) was used as the substrate. Chemicals Technical grade hexane and acetone were re-distilled from glass and the purity of the distillate checked by gas chromatography. All other reagents used were of analytical grade. Hexamethyldisilazane, trichloromethylsilane and nicotinamide were obtained from Koch-Light Ltd., Coinbrook, Bucks. Procedures After decapitation and exsanguination, the livers were excised and kept at 0-4°C for use within 3 hr. Body weights and organ weights were recorded. Washed liver microsomes were then prepared as described previously (Chipman et al., 1979), resuspended in 1.15~o KCI solution and
l 4 1 8
Male Female Male Male/Female Pool Female Pool Male 6
4
4
6
Male Female
7
6
Female
Male
4
Male 11
20
11
Male Female
7
12
Male Female
Number assayed
Sex
310 _ 30
810
230 221 ___ 7
436 362 _+ 15
1980 _+ 20
2500 _ 26
1250 +_ 55
1440 _+ 44
683 + 19
721 _ 18
487 + 14
523 _ 20
295 +_ 9
320 _+ 7
Body wt (g)
4.83 + 0.24
Specific activity bird
Specific activity male SD rat
x
Liver wt/body wt bird Liver wt/body wt male SD r a t
13.5 + 1.1
13.6
7.8 13.4
6.90 5.53 4- 0.47
16.6 _+ 2.73
10.9 _ 1.18
13.8 + 1.93
14.7 _+ 1.40
9.30 + 0.57
12.9 __+0.76
7.40 + 0.58
7.37 ___0.64
9.10 _ 1.61
6.53 _ 0.68
3.00 _+ 0.11
0.37
1.11 + 0.25 (0.24-2.70) 0.306 _+ 0.170 (0.053-1.200) 0.484 + 0.14 (0.170-1.510) 0.863 4- 0.151 (0.140-2.000) 0.690 -I- 0.240 (0.120-1.28) 0.958 _+ 0.177 (0.180-2.000) 1.24 _ 0.351 (0.30-2.20) 0.697 _ 0.228 (0.020-0.95) 0.333 4- 0.112 (0.110-0.74) 0.443 + 0.374 (0.062-I.19) 6.63 1.70 _+ 0.067 (0.21-3.2) 0.20 0.31
1.00 _ 0.04
0.057
0.268 _ 0.070 (0.0450-0.764) 0.103 ___ 0.036 (0.0114).330) 0.092 + 0.024 (0.037-0.258) 0.168 _ 0.028 (0.0284).368) 0.119 _ 0.040 (0.0184).212) 0.182 + 0.037 (0.0314).368) 0.226 _+ 0.061 (0.055--0.411) 0.138 _+ 0.045 (0.005--0.180) 0.057 4- 0.014 (0.019-0.104) 0.072 _+ 0.059 (0.0124).190) I. 12 0.28 _+ 0.11 (0.04-0.52) 0.040 0.066
Variability is given as + SEM throughout, although it should be noted that the distribution of activities was not Gaussian for some of the sea birds. For these, variability is also indicated by the ranges given in brackets below the means.
(2) Relative activity =
2.22
2.91 3.10 ___ 0.08
2.44 2.37 _+ 0.11
2.45 _ 0.061
3.00 _+ 0.49
2.80 _ 0.059
2.68 _ 0.10
2.65 + 0.12
2.46 ___0.13
2.82 _ 0.08
2.77 _ 0.12
2.85 + 0.15
3.27 _ 0.17
Liver wt/body wt _ SEM
(1) Specific activity as nmoles H E O M diol formed mg microsomal protein- ~ m i n - 1.
* Activities are expressed in two ways.
Rat (Sprague-Dawley)
(Larus argentatus)
Herring gull
(Larus ridibundus)
Black-headed gull
(Puffinus puffinus)
Manx shearwater
(Phalacrocorax carbo)
Cormorant
(Phalacrocorax aristotelis)
Shag
(Uria aaloe)
Guillemot
(Alca torda)
Razorbill
(Fratercula artica)
Puffin
Species
Epoxide hydrase activities* Microsomal (I) Specific protein nmol m g - ~ m i n - ~ (2) Relative mg g - ~ + SEM (range) (range)
Table 1. Epoxide hydrolase activities in different species
> I"
"r ,-q
7
Hepatic microsomal epoxide hydrolase in sea birds diluted as necessary for the determination of epoxide hydrolase activity. Epoxide hydrolase activity was determined as described previously (Walker et al., 1975) using conditions which gave linearity with respect to both time and protein concentration. Reaction medium (pH 7.4) 4.5 ml, containing 248 #mol sodium phosphate, 62.8 #mol KC1 and 25 pmol nicotinamide, was placed in a 25 ml Ehrlenmeyer flask, to which was added 0.5 ml aliquots of diluted microsomal suspension. After pre-incubation for 90 sec at either 37°C (mammals) or 42°C (birds) in a Mickle shaking water bath, the reaction was started by the addition of 20 #1 HEOM substrate solution. Incubation times were 15-30 min, and were terminated by extraction with diethyl ether (3 ml). Blank extractions were carried out to enable correction to be made, if necessary, for any background peaks. Extraction of substrate and metabolites was completed by two further ether partitions (2 x 3 ml). Trimethyl silyl (TMS) derivatives of HEOM metabolites were prepared as previously described (Walker & E1 Zorgani, 1974). These derivatized extracts were examined on either a Perkin Elmer F11 or a Pye 204 gas chromatograph fitted with a 63Ni electron capture detector and glass columns. The stationary phase used was 2~o w/w SE52 and 0.5~ w/w Epikote 1001 resin on 80-100 mesh AW-DMCS treated Chromosorb W. Cleaned extracts of brain were examined in a similar way, but employing an Apiezon column (Knight & Walker, 1982). Quantitative determinations were based on comparison of the peak areas of standards with those of unknown peaks. Microsomal protein was determined by the method of Lowry et al. (1951).
RESULTS The activities of epoxide hydrolase in different species are given in Table 1, expressed b o t h as specific activities a n d as relative activities (see footnote to Table). The m e a n values for specific activity fall into the range 0.306-1.70 for the six fish-eating species at the top of the Table. The single male M a n x shearwater gave a value of 6.63, the highest recorded in the study. A previous study upon H E O M - e p o x i d e hydrolase activity in birds gave a range of 0.0045-1.26 (Walker, 1976). Nearly all the values here fall within the upper part of this range. The two o m n i v o r o u s gulls gave values ranging from 0.20 to 0.37, which coincide with the lower end of the range for fisheating birds. Generally speaking these activities fall below the range for m a m m a l s especially if compari-
465
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4
--
3
--
•
•
•
i
E 2--
•
"~.1
•
I
05 Actiwty,
•
I
I.O
.I
5.0
tog score
Fig. 1. The distribution of epoxide hydrolase activities amongst female razorbills. Relative activities were determined as noted in Table 1. Activities are plotted on a log scale. The number of individuals having activity in a particular range are plotted against the log of the highest concentration in that range. son is based upon relative activities which take account of liver wei~,ht (Table 1 and Walker, 1980). Amongst the fish-eating birds there were no clear cut species or sex differences. Male puffins gave activities more than twice as high as female puffins, but this was not statistically significant. The nature of the variability in epoxide hydrolase was investigated further in the species for which most individuals were a s s a y e d - - t h e razorbill, puffin a n d guillemot. The general level of variability in activity was less than was found with microsomal mono-oxygenase. In female razorbills, for instance, there was a 14-fold difference in specific activity between the highest and the lowest individuals, c o m p a r e d with 21- a n d 58-fold differences in mono-oxygenase activity found by two contrasting assay procedures. In male puffins the epoxide hydrolase variability was l l-fold c o m p a r e d with figures of 65- and 19-fold in the case of the mono-oxygenase assays (Knight & Walker, 1982). The frequency diagram for relative epoxide hydrolase activity in the female razorbill (n = 20) is shown in Fig. 1. It reveals a peak of irregular shape, in contrast to the distribution found for microsomal mono-oxygenase in the same specimens (Knight & Walker, 1982), where more
Table 2. Epoxide hydrolase activities in liver homogenates and microsomes
Species Razorbill Guillemot Puffin Puffin Puffin Puffin
Sex
Microsomal protein yield mg protein g liver-1
Epoxide hydrolase activity in homogenate nmol g liver-1 min-1
Epoxide hydrolase activity in microsomal precipitate nmol g liver-~ min ]
Female Male Male Female Female Female
10.3 11.5 5.0 1 I. 1 10.4 11.5
24 33 23 160 124 145
11.3 9.1 7.2 0.59 1.8 2.6
Estimated ~o total epoxide hydrolase activity in microsomes 120 70 78 1.0 2.7 4.6
All values are uncorrected specific activities, and represent means for three or more replicates. * Assuming 40~ of total microsomal yield is recovered in microsomal precipitate (see Chipman & Walker, 1979).
466
G. C. KNIGHTand C. H. WALKER
Table 3. Epoxide hydrolase activities in different subcellular fractions of the livers in early season female puffins Sample Sample Sample 1 2 3
Fraction Homogenate 11,000 ,q precipitate Microsomal fraction 105,000 ,g supernatant
100 105 5.3 0
100 106 3.8 0
100 140 2.1 0
Activities expressed as a percentage of that present in the original homogenate. widely spread values for mono-oxygenase were arranged into two distinct activity peaks. With regard to epoxide hydrolase activity, male puffin (n = 12) showed a similar distribution and range to that found in the female razorbill, whereas female guillemots (n = 11) gave a distribution of narrower range which approximated to a simple Gaussian curve. The enzyme activity in original homogenates was compared with that in the microsomal fraction for certain specimens (Table 2). As can be seen, 31~7% of the epoxide hydrolase activity present in the homogenate was recovered in the microsomal precipitate in the case of one female razorbill, one male guillemot, and one male puffin. Previous experience with the preparation of rat microsomes by this procedure suggests that only some 40% of the original microsomal protein is actually recovered in the microsomal fraction so, following from this, it appears that > 70% of the epoxide hydrolase activity in these homogenates was actually located in the microsomes. By contrast, only a small proportion (< 2.6%) of the epoxide hydrolase in homogenates of three female puffins was found in the microsomal precipitate. Most of the mono-oxygenase activity was in the microsomal fraction in the case of all the specimens shown in Table 2. Subsequent work on the subcellular fractionation of liver homogenates of three female puffins showed that nearly all of the hydrolase activity came down at 11,000 g (Table 3). Little activity was found in the microsomes, and none at all in the 105,000 0 supernatant. It seems likely that this activity is associated with one of the organelles (e.g. mitochondria or lysosomes) which are found in this precipitate. Since protein yields were above average, it is unlikely that this O26O
+
0.80
•
+
o2c, a
0.64
._~
ll=
R o~- 0156 .¢:
048
" 0.104
-+ --
+
•
+
--
I
I
I
{
I ÷
L
2
4
6
8
I0
12
Time
.~
U
+
+
:
0.32
._R
0.16
0 14
of seoson, week
Fig. 2. Epoxide hydrolase activity of puffins in relation to time of breeding season. Relative activities (see Table 1) are plotted against the date upon which the assay was performed. On the scale of weeks 4 = 20 May, 8 = 17 June, 12 = 15 July.
result is due to "heavy" microsomes being sedimented at low 9. As with the study of mono-oxygenase activity reported earlier, the data was examined for any relationship between enzyme activity and geographical location or time of season (Knight & Walker, 1982). Males and females were analysed separately in the case of razorbill, puffin, guillemot, cormorant and shag. The relationship between enzyme activity and PCB levels (1978 and 1979 samples only) was also investigated but in puffin and razorbill only, taking the whole sample (both sexes) for each species. No clear correlations were found for any of these combinations except that of enzyme activity vs time of season in the female puffin (Fig. 2). As can be seen, four early-season birds all showed low activities with a steady rise of activity in the samples taken later in the season. Although the sample is small, the distinction between the two groups is clear cut and appears to be real. These activities were not correlated with the size of ovaries. In the case of male puffins, female razorbills and female guillemots, the mono-oxygenase activity (measured with either aldrin or HCE) was plotted against the hydrolase activity for each specimen. No correlation was found between epoxide hydrolase activity and mono-oxygenase activity in any of these three groups.
DISCUSSION
These results provide further evidence of broad phylogenetic differences in epoxide hydrolase activity determined with HEOM following the trend mammals > birds > fish (Walker, 1980). On limited evidence a similar trend is found with benzo(a)pyrene 4,5 oxide substrate, but not with styrene oxide substrate, suggesting the existence of different forms of the enzyme in the different groups (Walker et al., 1975). In contrast to the mono-oxygenase activities of these species, these values fall within the general range for birds. Mono-oxygenase activities tended to be substantially lower in fish-eating birds than in other birds, lending support to the idea that certain forms of the enzyme may have evolved to facilitate the detoxication of naturally-occurring xenobiotics in herbivores and omnivores (Walker, 1980). No such general proposition can be advanced for HEOMepoxide hydrolase either in theory or on the grounds of present evidence. In the present study epoxide hydrolase also contrasted with mono-oxygenase in respect of intraspecific variability. Frequency diagrams for epoxide hydrolase do not always show simple Gaussian distributions, but neither do they show the widely spread multiple peaks found for mono-oxygenase in the razorbill and puffin (Knight & Walker, 1982). Apart from the seasonal variation in the female puffin discussed above, no relationship was found between epoxide hydrolase activity and time of season, geographical location, or PCB residues in any of the sex/species combinations studied. It should be emphasized, however, that this conclusion can only be a tentative one because of the small numbers of individuals involved. Only the razorbills (both sexes) and male puffins and female guillemots
Hepatic microsomal epoxide hydrolase in sea birds were represented by 10 or more individuals. In most other cases the n u m b e r s were too small to show anything other than a major trend in the data. The a p p a r e n t increase of microsomal epoxide hydrolase activity in female puffins during the breeding season deserves further comment. T h e early season females showed low activity in microsomes but high activity in the 11,000 g precipitate. This suggests that the location of epoxide hydrolase within the cell may be dependent upon season. The activity was not clearly related to the size of the ovaries, so it is uncertain whether this seasonal change is related to reproduction. In view of the role of epoxide hydrolase in metabolizing potentially harmful epoxides generated by the mono-oxygenase it is interesting to compare the activities of these two enzyme systems. Referring to the mono-oxygenase activities previously determined upon these specimens (Knight & Walker, 1982) it is interesting to note that female puffins, and to a lesser extent male puffins show high mono-oxygenase activities in relation to epoxide hydrolase activities when c o m p a r e d with the other species. The possibility should therefore be considered that puffins may be less effective than the other species in detoxifying epoxides generated by the endoplasmic reticulum of the liver. Acknowledyements--This work was supported by an NERC grant. The authors are grateful to Dr D. C. Cabot, Dr M. Harris, Dr C. Lloyd, Dr G. Westlake, Dr P. Stanley, Dr D. Osborn, Mr M. Tasker and Mr S. Murray for their help in obtaining specimens, and to Mr S. Hallam and Mr J. Horne for technical assistance.
REFERENCES BINDEL U., SPARROW A. SCHMASSMANN U., GOLAN M., BENTLEY P. t~ OESCH P. 0979) Endogenous role of epoxide hydratase. Eur. J. Biochem. 97, 275-281. BROOKS G. T., HARRISON A. t~ LEWIS S. E. (1970) Cyclodiene epoxide ring hydration by microsomes from mam-
467
malian liver and houseflies. Biochem. Pharmac. 19, 255-273. CmPMAN J. K., KURUKGY M. & WALKER C. H. (1979) Comparative metabolism of a dieldrin analogue; Hepatic microsomal systems as models for metabolism in the whole animal. Biochem. Pharmac. 28, 69-75. CHIPMAN J. K. & WALKER C. H. (1979) The metabolism of HEOD and two of its analogues: The relationship between rates of metabolism and rates of excretion of metabolites in the male rat. Biochem. Pharmac. 28, 1337-1345. CRAVEN A. C. C. (1977) A comparative study of vertebrate epoxide hydratase using HEOM as substrate. Ph.D. Thesis, Reading University. CRAVEN A. C. C., BROOKS G. T. & WALKER C. H. (1976) The inhibition of HEOM epoxide hydrase in mammalian liver microsomes and insect pupal homogenates. Pestic. Biochem. Physiol. 6, 132-141. KNIGHT G. C. & WALKER C. H. (1982) A study of the hepatic microsomal mono-oxygenase of sea birds and its relationship to organochlorine pollutants. Comp. Biochem. Physiol. 73C, 211 221. LOWRY O. H., ROSEBROUGHN. J., FARR A. L. & RANDALL R. J. (1951) Protein measurements with the Folin phenol reagent. J. biol. Chem. 193, 265-275. OESCH F. (1973) Mammalian epoxide hydrases: inducible enzymes catalysing the inactivation of carcinogenic and cytotoxic metabolites derived from aromatic and olefinic compounds. Xenobiotica 3, 305-340. WALKER C. H. 0976) The significance of pesticide residues in the environment. Outlook Ayric. 9, 16-20. WALKER C. H. (1980) Species variations in some enzymes that metabolize xenobiotics. Pro(]. Dru9 Metab. 5, 113-164. WALKER C. H., CRAVEN A. C. C. & KURUKGY M. (1975) The metabolism of organochlorine compounds by microsomal enzymes of the shag. Envir. Physiol. Biochem. 5, 58 64. WALKER C. H. & EL ZORGANI G. A. (1974) The comparative metabolism and excretion of HCE, a biodegradable analogue of dieldrin, by vertebrate species. Archs envir. contam. Toxicol. 1, 97-116. WILKINSON C. F. (1976) Insecticide Biochemistry and Physiology (Edited by WILKINSON C. F.}. Plenum Press, New York.
0306-4492/82/060463-05503.00/0 © 1982 Pergamon Press Ltd
A S T U D Y O F THE H E P A T I C M I C R O S O M A L H Y D R O L A S E IN SEA BIRDS
EPOXIDE
G. C. KNIGHT and C. H. WALKER Department of Physiology and Biochemistry, The University, Whiteknights, Reading, Berks. RG6 2A J, UK (Received 3 March 1982) Abstract--l. Epoxide hydrolase activities were measured in six species of sea birds using the chlorinated epoxide HEOM as substrate. 2. The activities found fell within the general range for birds, and there were no clear sex or species differences. 3. Relatively low activities were found in liver microsomes of female puffins early in the breeding season, but high activities were found in 11,000 g precipitates from these individuals. Apart from this no correlations were found between activity and time of year, PCB residues or geographical location. 4. These results are discussed in relation to mono-oxygenase activities in the same samples.
INTRODUCTION Hepatic microsomal epoxide hydrolase can convert a wide range of exogenous and endogenous epoxides into trans diols. Substrates of the enzyme include the epoxides of polycyclic aromatic h y d r o c a r b o n s a n d steroids (Oesch, 1973; Bindel et al., 1979), insect juvenile h o r m o n e s and analogues thereof (see Wilkinson, 1976), and chlorinated insecticides such as dieldrin (Brooks et al., 1970). An i m p o r t a n t role of the enzyme is the hydration of epoxides generated in the endoplasmic reticulum by the action of cytochrome P4s0-dependent mono-oxygenase upon unsaturated substrates. In this way the cell may be protected against the toxic action of certain active oxides (e.g. of polycyclic a r o m a t i c hydrocarbons) which are formed in vivo. The enzyme is clearly of interest in connection with pollution problems. Apart from the question of the deactivation of potentially harmful epoxides referred to above, epoxide hydrolase attack can provide an effective mechanism for elimination of certain substances which are liable to undergo bioaccumulation. Thus, trans diols formed from dieldrin or its analogues, or from epoxides of polychlorinated biphenyls generated in situ, are readily excreted, usually as conjugates. The present investigation was concerned with the activity of this enzyme in fish-eating sea birds, a group with a m a r k e d tendency to bioaccumulate liposoluble pollutants. The status of hepatic microsomal mono-oxygenase was studied in the same specimens, and these results are reported elsewhere (Knight & Walker, 1982). MATERIALS AND METHODS Animals All the birds used in this study were adults. The sea birds were of the following species: the puffin (Fratercula arctica), razorbill (Alca torda) and guillemot (Uria aalge) of * HEOM = 1,2,3,4,9,9-hexachloro-l,4,4a,5,6,7,8,8a-octahydro-6,7,-epoxy- 1A-methanonaphthalene. 463
the family Alcidae, order Charadriformes; the shag (Phalacrocorax aristotelis) and cormorant (Phalacrocorax carbo) of the family Phalacrocoracidae, order Pelecaniformes; and the Manx shearwater (Puffinus puffinus) of the family Procellariidae, order Procellariiformes. Collections were made, under licence, at approximately one month intervals during the period May-July of 1978-1980 from each of the following sites: The Saltee Islands, Co. Wexford, Eire (52 ° 7'N. 6 ° 35'W) and the Isle of May, Fife, Scotland (56 ° l l'N, 2 ° 33'W). A single collection was also made from St. Kilda, Outer Hebrides, Scotland (57 ° 50'N, 8 ° 40'W) in June 1979. The puffin and Manx shearwater were the only species taken from St. Kilda; all cormorants came from the Saltee Islands. After capture in the breeding colonies, the birds were transported back to the laboratory in large, well-ventilated plastic containers, and maintained on a diet of sprats (Sprattus sprattus), with an adequate supply of water, until used for experimentation (usually within 5 days of capture). Cormorants were frequently maintained for longer periods. Adult male Sprague-Dawley rats (300400g) were maintained as previously described (Chipman & Walker, 1979). Rooks (Corvus frugilegus) and black headed gulls (Larus ridibundus) were obtained from the Pest Infestation Control Laboratory, Ministry of Agriculture, Fisheries and Food, Tolworth, Surrey and had been caught in the wild. Substrate and metabolite HEOM* and its metabolite HEOM trans-diol were prepared as previously described (Craven et al., 1976; Craven, 1977). HEOM in ethanolic solution (2 mg ml-1) was used as the substrate. Chemicals Technical grade hexane and acetone were re-distilled from glass and the purity of the distillate checked by gas chromatography. All other reagents used were of analytical grade. Hexamethyldisilazane, trichloromethylsilane and nicotinamide were obtained from Koch-Light Ltd., Coinbrook, Bucks. Procedures After decapitation and exsanguination, the livers were excised and kept at 0-4°C for use within 3 hr. Body weights and organ weights were recorded. Washed liver microsomes were then prepared as described previously (Chipman et al., 1979), resuspended in 1.15~o KCI solution and
l 4 1 8
Male Female Male Male/Female Pool Female Pool Male 6
4
4
6
Male Female
7
6
Female
Male
4
Male 11
20
11
Male Female
7
12
Male Female
Number assayed
Sex
310 _ 30
810
230 221 ___ 7
436 362 _+ 15
1980 _+ 20
2500 _ 26
1250 +_ 55
1440 _+ 44
683 + 19
721 _ 18
487 + 14
523 _ 20
295 +_ 9
320 _+ 7
Body wt (g)
4.83 + 0.24
Specific activity bird
Specific activity male SD rat
x
Liver wt/body wt bird Liver wt/body wt male SD r a t
13.5 + 1.1
13.6
7.8 13.4
6.90 5.53 4- 0.47
16.6 _+ 2.73
10.9 _ 1.18
13.8 + 1.93
14.7 _+ 1.40
9.30 + 0.57
12.9 __+0.76
7.40 + 0.58
7.37 ___0.64
9.10 _ 1.61
6.53 _ 0.68
3.00 _+ 0.11
0.37
1.11 + 0.25 (0.24-2.70) 0.306 _+ 0.170 (0.053-1.200) 0.484 + 0.14 (0.170-1.510) 0.863 4- 0.151 (0.140-2.000) 0.690 -I- 0.240 (0.120-1.28) 0.958 _+ 0.177 (0.180-2.000) 1.24 _ 0.351 (0.30-2.20) 0.697 _ 0.228 (0.020-0.95) 0.333 4- 0.112 (0.110-0.74) 0.443 + 0.374 (0.062-I.19) 6.63 1.70 _+ 0.067 (0.21-3.2) 0.20 0.31
1.00 _ 0.04
0.057
0.268 _ 0.070 (0.0450-0.764) 0.103 ___ 0.036 (0.0114).330) 0.092 + 0.024 (0.037-0.258) 0.168 _ 0.028 (0.0284).368) 0.119 _ 0.040 (0.0184).212) 0.182 + 0.037 (0.0314).368) 0.226 _+ 0.061 (0.055--0.411) 0.138 _+ 0.045 (0.005--0.180) 0.057 4- 0.014 (0.019-0.104) 0.072 _+ 0.059 (0.0124).190) I. 12 0.28 _+ 0.11 (0.04-0.52) 0.040 0.066
Variability is given as + SEM throughout, although it should be noted that the distribution of activities was not Gaussian for some of the sea birds. For these, variability is also indicated by the ranges given in brackets below the means.
(2) Relative activity =
2.22
2.91 3.10 ___ 0.08
2.44 2.37 _+ 0.11
2.45 _ 0.061
3.00 _+ 0.49
2.80 _ 0.059
2.68 _ 0.10
2.65 + 0.12
2.46 ___0.13
2.82 _ 0.08
2.77 _ 0.12
2.85 + 0.15
3.27 _ 0.17
Liver wt/body wt _ SEM
(1) Specific activity as nmoles H E O M diol formed mg microsomal protein- ~ m i n - 1.
* Activities are expressed in two ways.
Rat (Sprague-Dawley)
(Larus argentatus)
Herring gull
(Larus ridibundus)
Black-headed gull
(Puffinus puffinus)
Manx shearwater
(Phalacrocorax carbo)
Cormorant
(Phalacrocorax aristotelis)
Shag
(Uria aaloe)
Guillemot
(Alca torda)
Razorbill
(Fratercula artica)
Puffin
Species
Epoxide hydrase activities* Microsomal (I) Specific protein nmol m g - ~ m i n - ~ (2) Relative mg g - ~ + SEM (range) (range)
Table 1. Epoxide hydrolase activities in different species
> I"
"r ,-q
7
Hepatic microsomal epoxide hydrolase in sea birds diluted as necessary for the determination of epoxide hydrolase activity. Epoxide hydrolase activity was determined as described previously (Walker et al., 1975) using conditions which gave linearity with respect to both time and protein concentration. Reaction medium (pH 7.4) 4.5 ml, containing 248 #mol sodium phosphate, 62.8 #mol KC1 and 25 pmol nicotinamide, was placed in a 25 ml Ehrlenmeyer flask, to which was added 0.5 ml aliquots of diluted microsomal suspension. After pre-incubation for 90 sec at either 37°C (mammals) or 42°C (birds) in a Mickle shaking water bath, the reaction was started by the addition of 20 #1 HEOM substrate solution. Incubation times were 15-30 min, and were terminated by extraction with diethyl ether (3 ml). Blank extractions were carried out to enable correction to be made, if necessary, for any background peaks. Extraction of substrate and metabolites was completed by two further ether partitions (2 x 3 ml). Trimethyl silyl (TMS) derivatives of HEOM metabolites were prepared as previously described (Walker & E1 Zorgani, 1974). These derivatized extracts were examined on either a Perkin Elmer F11 or a Pye 204 gas chromatograph fitted with a 63Ni electron capture detector and glass columns. The stationary phase used was 2~o w/w SE52 and 0.5~ w/w Epikote 1001 resin on 80-100 mesh AW-DMCS treated Chromosorb W. Cleaned extracts of brain were examined in a similar way, but employing an Apiezon column (Knight & Walker, 1982). Quantitative determinations were based on comparison of the peak areas of standards with those of unknown peaks. Microsomal protein was determined by the method of Lowry et al. (1951).
RESULTS The activities of epoxide hydrolase in different species are given in Table 1, expressed b o t h as specific activities a n d as relative activities (see footnote to Table). The m e a n values for specific activity fall into the range 0.306-1.70 for the six fish-eating species at the top of the Table. The single male M a n x shearwater gave a value of 6.63, the highest recorded in the study. A previous study upon H E O M - e p o x i d e hydrolase activity in birds gave a range of 0.0045-1.26 (Walker, 1976). Nearly all the values here fall within the upper part of this range. The two o m n i v o r o u s gulls gave values ranging from 0.20 to 0.37, which coincide with the lower end of the range for fisheating birds. Generally speaking these activities fall below the range for m a m m a l s especially if compari-
465
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4
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3
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i
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•
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05 Actiwty,
•
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I.O
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5.0
tog score
Fig. 1. The distribution of epoxide hydrolase activities amongst female razorbills. Relative activities were determined as noted in Table 1. Activities are plotted on a log scale. The number of individuals having activity in a particular range are plotted against the log of the highest concentration in that range. son is based upon relative activities which take account of liver wei~,ht (Table 1 and Walker, 1980). Amongst the fish-eating birds there were no clear cut species or sex differences. Male puffins gave activities more than twice as high as female puffins, but this was not statistically significant. The nature of the variability in epoxide hydrolase was investigated further in the species for which most individuals were a s s a y e d - - t h e razorbill, puffin a n d guillemot. The general level of variability in activity was less than was found with microsomal mono-oxygenase. In female razorbills, for instance, there was a 14-fold difference in specific activity between the highest and the lowest individuals, c o m p a r e d with 21- a n d 58-fold differences in mono-oxygenase activity found by two contrasting assay procedures. In male puffins the epoxide hydrolase variability was l l-fold c o m p a r e d with figures of 65- and 19-fold in the case of the mono-oxygenase assays (Knight & Walker, 1982). The frequency diagram for relative epoxide hydrolase activity in the female razorbill (n = 20) is shown in Fig. 1. It reveals a peak of irregular shape, in contrast to the distribution found for microsomal mono-oxygenase in the same specimens (Knight & Walker, 1982), where more
Table 2. Epoxide hydrolase activities in liver homogenates and microsomes
Species Razorbill Guillemot Puffin Puffin Puffin Puffin
Sex
Microsomal protein yield mg protein g liver-1
Epoxide hydrolase activity in homogenate nmol g liver-1 min-1
Epoxide hydrolase activity in microsomal precipitate nmol g liver-~ min ]
Female Male Male Female Female Female
10.3 11.5 5.0 1 I. 1 10.4 11.5
24 33 23 160 124 145
11.3 9.1 7.2 0.59 1.8 2.6
Estimated ~o total epoxide hydrolase activity in microsomes 120 70 78 1.0 2.7 4.6
All values are uncorrected specific activities, and represent means for three or more replicates. * Assuming 40~ of total microsomal yield is recovered in microsomal precipitate (see Chipman & Walker, 1979).
466
G. C. KNIGHTand C. H. WALKER
Table 3. Epoxide hydrolase activities in different subcellular fractions of the livers in early season female puffins Sample Sample Sample 1 2 3
Fraction Homogenate 11,000 ,q precipitate Microsomal fraction 105,000 ,g supernatant
100 105 5.3 0
100 106 3.8 0
100 140 2.1 0
Activities expressed as a percentage of that present in the original homogenate. widely spread values for mono-oxygenase were arranged into two distinct activity peaks. With regard to epoxide hydrolase activity, male puffin (n = 12) showed a similar distribution and range to that found in the female razorbill, whereas female guillemots (n = 11) gave a distribution of narrower range which approximated to a simple Gaussian curve. The enzyme activity in original homogenates was compared with that in the microsomal fraction for certain specimens (Table 2). As can be seen, 31~7% of the epoxide hydrolase activity present in the homogenate was recovered in the microsomal precipitate in the case of one female razorbill, one male guillemot, and one male puffin. Previous experience with the preparation of rat microsomes by this procedure suggests that only some 40% of the original microsomal protein is actually recovered in the microsomal fraction so, following from this, it appears that > 70% of the epoxide hydrolase activity in these homogenates was actually located in the microsomes. By contrast, only a small proportion (< 2.6%) of the epoxide hydrolase in homogenates of three female puffins was found in the microsomal precipitate. Most of the mono-oxygenase activity was in the microsomal fraction in the case of all the specimens shown in Table 2. Subsequent work on the subcellular fractionation of liver homogenates of three female puffins showed that nearly all of the hydrolase activity came down at 11,000 g (Table 3). Little activity was found in the microsomes, and none at all in the 105,000 0 supernatant. It seems likely that this activity is associated with one of the organelles (e.g. mitochondria or lysosomes) which are found in this precipitate. Since protein yields were above average, it is unlikely that this O26O
+
0.80
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0.64
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048
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+
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I
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L
2
4
6
8
I0
12
Time
.~
U
+
+
:
0.32
._R
0.16
0 14
of seoson, week
Fig. 2. Epoxide hydrolase activity of puffins in relation to time of breeding season. Relative activities (see Table 1) are plotted against the date upon which the assay was performed. On the scale of weeks 4 = 20 May, 8 = 17 June, 12 = 15 July.
result is due to "heavy" microsomes being sedimented at low 9. As with the study of mono-oxygenase activity reported earlier, the data was examined for any relationship between enzyme activity and geographical location or time of season (Knight & Walker, 1982). Males and females were analysed separately in the case of razorbill, puffin, guillemot, cormorant and shag. The relationship between enzyme activity and PCB levels (1978 and 1979 samples only) was also investigated but in puffin and razorbill only, taking the whole sample (both sexes) for each species. No clear correlations were found for any of these combinations except that of enzyme activity vs time of season in the female puffin (Fig. 2). As can be seen, four early-season birds all showed low activities with a steady rise of activity in the samples taken later in the season. Although the sample is small, the distinction between the two groups is clear cut and appears to be real. These activities were not correlated with the size of ovaries. In the case of male puffins, female razorbills and female guillemots, the mono-oxygenase activity (measured with either aldrin or HCE) was plotted against the hydrolase activity for each specimen. No correlation was found between epoxide hydrolase activity and mono-oxygenase activity in any of these three groups.
DISCUSSION
These results provide further evidence of broad phylogenetic differences in epoxide hydrolase activity determined with HEOM following the trend mammals > birds > fish (Walker, 1980). On limited evidence a similar trend is found with benzo(a)pyrene 4,5 oxide substrate, but not with styrene oxide substrate, suggesting the existence of different forms of the enzyme in the different groups (Walker et al., 1975). In contrast to the mono-oxygenase activities of these species, these values fall within the general range for birds. Mono-oxygenase activities tended to be substantially lower in fish-eating birds than in other birds, lending support to the idea that certain forms of the enzyme may have evolved to facilitate the detoxication of naturally-occurring xenobiotics in herbivores and omnivores (Walker, 1980). No such general proposition can be advanced for HEOMepoxide hydrolase either in theory or on the grounds of present evidence. In the present study epoxide hydrolase also contrasted with mono-oxygenase in respect of intraspecific variability. Frequency diagrams for epoxide hydrolase do not always show simple Gaussian distributions, but neither do they show the widely spread multiple peaks found for mono-oxygenase in the razorbill and puffin (Knight & Walker, 1982). Apart from the seasonal variation in the female puffin discussed above, no relationship was found between epoxide hydrolase activity and time of season, geographical location, or PCB residues in any of the sex/species combinations studied. It should be emphasized, however, that this conclusion can only be a tentative one because of the small numbers of individuals involved. Only the razorbills (both sexes) and male puffins and female guillemots
Hepatic microsomal epoxide hydrolase in sea birds were represented by 10 or more individuals. In most other cases the n u m b e r s were too small to show anything other than a major trend in the data. The a p p a r e n t increase of microsomal epoxide hydrolase activity in female puffins during the breeding season deserves further comment. T h e early season females showed low activity in microsomes but high activity in the 11,000 g precipitate. This suggests that the location of epoxide hydrolase within the cell may be dependent upon season. The activity was not clearly related to the size of the ovaries, so it is uncertain whether this seasonal change is related to reproduction. In view of the role of epoxide hydrolase in metabolizing potentially harmful epoxides generated by the mono-oxygenase it is interesting to compare the activities of these two enzyme systems. Referring to the mono-oxygenase activities previously determined upon these specimens (Knight & Walker, 1982) it is interesting to note that female puffins, and to a lesser extent male puffins show high mono-oxygenase activities in relation to epoxide hydrolase activities when c o m p a r e d with the other species. The possibility should therefore be considered that puffins may be less effective than the other species in detoxifying epoxides generated by the endoplasmic reticulum of the liver. Acknowledyements--This work was supported by an NERC grant. The authors are grateful to Dr D. C. Cabot, Dr M. Harris, Dr C. Lloyd, Dr G. Westlake, Dr P. Stanley, Dr D. Osborn, Mr M. Tasker and Mr S. Murray for their help in obtaining specimens, and to Mr S. Hallam and Mr J. Horne for technical assistance.
REFERENCES BINDEL U., SPARROW A. SCHMASSMANN U., GOLAN M., BENTLEY P. t~ OESCH P. 0979) Endogenous role of epoxide hydratase. Eur. J. Biochem. 97, 275-281. BROOKS G. T., HARRISON A. t~ LEWIS S. E. (1970) Cyclodiene epoxide ring hydration by microsomes from mam-
467
malian liver and houseflies. Biochem. Pharmac. 19, 255-273. CmPMAN J. K., KURUKGY M. & WALKER C. H. (1979) Comparative metabolism of a dieldrin analogue; Hepatic microsomal systems as models for metabolism in the whole animal. Biochem. Pharmac. 28, 69-75. CHIPMAN J. K. & WALKER C. H. (1979) The metabolism of HEOD and two of its analogues: The relationship between rates of metabolism and rates of excretion of metabolites in the male rat. Biochem. Pharmac. 28, 1337-1345. CRAVEN A. C. C. (1977) A comparative study of vertebrate epoxide hydratase using HEOM as substrate. Ph.D. Thesis, Reading University. CRAVEN A. C. C., BROOKS G. T. & WALKER C. H. (1976) The inhibition of HEOM epoxide hydrase in mammalian liver microsomes and insect pupal homogenates. Pestic. Biochem. Physiol. 6, 132-141. KNIGHT G. C. & WALKER C. H. (1982) A study of the hepatic microsomal mono-oxygenase of sea birds and its relationship to organochlorine pollutants. Comp. Biochem. Physiol. 73C, 211 221. LOWRY O. H., ROSEBROUGHN. J., FARR A. L. & RANDALL R. J. (1951) Protein measurements with the Folin phenol reagent. J. biol. Chem. 193, 265-275. OESCH F. (1973) Mammalian epoxide hydrases: inducible enzymes catalysing the inactivation of carcinogenic and cytotoxic metabolites derived from aromatic and olefinic compounds. Xenobiotica 3, 305-340. WALKER C. H. 0976) The significance of pesticide residues in the environment. Outlook Ayric. 9, 16-20. WALKER C. H. (1980) Species variations in some enzymes that metabolize xenobiotics. Pro(]. Dru9 Metab. 5, 113-164. WALKER C. H., CRAVEN A. C. C. & KURUKGY M. (1975) The metabolism of organochlorine compounds by microsomal enzymes of the shag. Envir. Physiol. Biochem. 5, 58 64. WALKER C. H. & EL ZORGANI G. A. (1974) The comparative metabolism and excretion of HCE, a biodegradable analogue of dieldrin, by vertebrate species. Archs envir. contam. Toxicol. 1, 97-116. WILKINSON C. F. (1976) Insecticide Biochemistry and Physiology (Edited by WILKINSON C. F.}. Plenum Press, New York.