Arch. Environ. Contain. Toxicol. 1 5 , 2 0 7 - 2 1 3 (1986)

nviron~| EA•hm• contamination of

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and

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9 I986 Springer-VerlagNew York Inc,

Metabolites of the Naphthenic Hydrocarbon Dodecylcyclohexane in Rainbow Trout Liver and Their Incorporation into Lipids J-E Cravedi and J. Tulliez Laboratoire de R e c h e r c h e s sur les Additifs Alimentaires, Institut National de la R e c h e r c h e Agronomique, B.R 3, 31931 Toulouse Cedex, France

Abstract. Rainbow trout (Salmo gairdneri R.) were force-fed 0.1 mCi (0.5 rag) 3H-dodecylcyclohexane and sacrificed at 12, 24, 48 hr and 1 week after feeding in order to study the metabolic utilization of a naphthenic hydrocarbon. One week after dosing, approximately 25% of the ingested radioactivity was stored in the carcass and 3/4of this radioactivity was due to unchanged hydrocarbon. In the liver, only 25% of the 3H present after one week was associated with hydrocarbon. The incorporation of radioactivity in hepatic lipids 24 hr after ingestion of 3H-dodecylcyclohexane revealed that radioactivity was equally incorporated into the phospholipids and neutral lipids. In neutral lipids, the free fatty acids were the most labeled fraction, whereas in phospholipids the greater deposition of radioactivity occurred in phosphatidyl choline. The major biotransformation products were characterized in the liver by thin layer chromatography, radio-gas chromatography and mass spectrometric analysis. Four metabolites, resulting from the oxidation of the alkyl chain or of the cyclohexane ring were identified; namely, 3-dodecylcyclohexanol, 4-dodecylcyclohexanot, cyclohexyldodecane-2-ol, and cyclohexyldodecanoic acid. This latter metabolite accounted for 30% of the liver radioactivity 24 hr after dosing. The toxicological relevance of this pathway is discussed.

Crude oils contain naphthenic hydrocarbons normally within a range of 15 to 40%, mainly in the form of 1-ring cycloparaffins (Posthuma I977;

Korte and Boedefeld 1978). In spite of these proportions and of the extensive existing studies relevant to petroleum hydrocarbon contamination of the environment, the fate of naphthenic hydrocarbons has received little attention, possibly because of their relatively low biological reactivity. Available data concern the accumulation of these compounds in various animal species (Tulliez and Bories 1975; Lawler et aI. 1978; Cravedi and Tulliez 1982a). However, in addition to the knowledge of the location and concentration of these hydrocarbons, it is necessary to know the nature and the amount of metabolites which are formed to ensure both protection of organisms and a safe food supply for man. The understanding of the metabolic pathways of naphthenic hydrocarbons in fish is all the more important as recently Luquet et at. (1984) reported that such compounds, precisely dodecylcyciohexane, r e d u c e d c o n s i d e r a b l y the growth of rainbow trout. The hypothesis of a possible metabolic effect of biotransformation products was put forward by these authors to explain the phenomenon. These observations led us to investigate the fate of cycloparaffins in Salmo gairdneri R. using 3H radiolabeled dodecylcyclohexane. Since liver plays a major role in the biotransformation of alkanes in rainbow trout (Cravedi and Tulliez, in press), emphasis has been placed on the characterization of metabolites isolated from hepatic tissue. Moreover, because unusual fatty acids such as cyclohexytdodecanoic acid may result from the m-oxidation of the alkyl chain (Cravedi and Tulliez 1982b), incorporation of metabolites into the lipid compartment in particular was investigated.

208

Material and Methods

J-E Cravedi and J. Tulliez were separated into different components as described by Tulliez and Bories (1978).

Radiochemicals 3H-dodecylcyclohexane was prepared from phenyldodecane which was labeled by direct contact with tritium gas (Wilzbach method) before hydrogenation in an autocalve at 10 atm in the presence of Adam's catalyst (Tulliez and Bories 1979). 3H-dodecylcyclohexane was purified by thin layer chromatography (TLC) using silica gel plates (Kieselge160, Merck) and hexane as the solvent. Its specific activity was 116 mCi/mM and radiochemical purity, as evaluated by radio-gas chromatography (radioGC) and radio-high performance liquid chromatography (radioHPLC) was >98%. 3H-dodecylcyclohexane was dissolved, in nonradioactive dodecylcyclohexane to a final specific activity of 20 txCi/mg.

Animals Rainbow trout (200-250 g) were obtained from the Institut National de la Recherche Agronomique pisciculture at Donzacq (Landes, France) and held in flowing dechlorinated tap water at 12~ for at least one week before use. The pH was constant at 7.3 and the photoperiod was 12 hr light (0600 to 1800 hr) and 12 hr dark. Once a day until 48 hr prior to an experiment, animals were fed ad libitum a pelleted commercial fish diet (Aqualim, France).

Experimental Twelve fish were anesthetized in a 0.05% (v/v) solution of 2phenoxyethanol (Koch-light Lab. Ltd) and force-fed a gelatin capsule containing 100 mg feed and 5 mg of 3H-dodecylcyclohexane (0,1 mCi). Immediately after dosing, trout were transferred to individual 30 L aquaria supplied with freshwater (4L/ rain). Groups of three animals each were killed by cervical dislocation after 12, 24, 48 hr, and 1 week for analyses.

Distribution Analyses Liver was excised from the fish, weighed, blotted and homogenized by means of a Polytron homogenizer at room temperature. The same procedure was used for the remaining carcasses, after careful removal of the gastro intestinal contents. One hundred mg of the homogenate was used for total measurement of radioactivity in tissues by combustion in an oxidizer (Intertechnique Oxymat) and subsequent liquid scintillation counting. These measurements were made in duplicate for each tissue, the rest of the homogenate was used for the isolation of total lipids, according to Folch's procedure (1957). Radioactivity in the residues after lipid extraction was measured by combustion as described above. Non-lipids refers to the upper phase of Folchwash of lipid extract plus residual non-extractable material. An aliquot of 24-hr total lipids was separated into particular classes on a silica gel column (Kieselgel 60, Merck) by successive hexane, chloroform, acetone, and methanol elutions, consisting of hydrocarbons, neutral lipids, glycolipidsand phosphatides fractions, respectively. Neutral lipids and phospholipids

Metabolite Isolation Purification and identification of metabolites were made on an aliquot of total lipids from 24 hr samples. Lipids were saponifled with ethanolic KOH and the unsaponifiable fraction was extracted with hexane. Fatty acids were extracted with petroleum ether from the aqueous phase after acidification by HC1, The extracts were concentrated and subjected to TLC in the solvent systems described in Table 5.

Analytical Procedures Derivatization: Fatty acids were esterified according to Cravedi and Tulliez (1982b). For acetylation, the extracts were evaporated in vacuo at 40~ and the residues were dissolved in acetic anhydride/pyridine (4:1, v/v), and kept at ambient temperature for 12 hr. Trimethylsilyl (TMS) derivatives were prepared by treating the various compounds with BSTFA (Pierce Chemical Co., Rockford, IL) in ethyl acetate for 4 hr at 70~ Reference Compounds: The following reference compounds were purchased from Fluka AG (Buchs, Switzerland): 4-Ethylcyclohexanol, 2-cyclohexylethanol, 2-hydroxyphenylacetic acid, 3-hydroxyphenylacetic acid and 4-hydroxyphenylacetic acid. 2-Hydroxycyclohexylacetic acid, 3-hydroxycyclohexylacetic acid and 4-hydroxycyclohexylacetic acid were prepared by catalytic hydrogenation of 2-hydroxyphenylacetic acid, 3-hydroxyphenylacetic acid and 4-hydroxyphenylacetic acid, respectively and purified by TLC.

TLC: Precoated silica gel glass plates (60, 250 ~, Merck) were used with the solvent system described above. Radioactive areas were located with a radiochromatogram scanner (II/LB 2723, Berthold). Radio-GC: A Girdel model 3000 gas chromatograph (GiravionsDorand, France) equipped with a radioactivity monitor (RGC 170 Perkin Elmer), flame-ionization detector, and a 1.5 m • 3.2 mm i.d. stainless steel column packed with 10% DEGS on 80/100 Supelcoport| was used.

GC/MS: A Hewlett Packard model 5992B gas chromatograph/ mass spectrometer equipped with a 12.5 m • 0.3 mm OV1 capillary column (Hewlen Packard) was used from 150~ to 250~ at 10~ and with a He (carrier gas) flow rate of 1 ml/min. Radiometric Assay: Radioactivity was measured in the various samples by liquid scintillation counting (Intertechnique SL32 apparatus).

Results Distribution o f Radioactivity The metabolic fate of naphthenic hydrocarbon was i n v e s t i g a t e d a f t e r a s i n g l e per os a d m i n i s t r a t i o n o f 5

Naphthenic Hydrocarbons in Trout Liver mg of 3H-dodecylcyclohexane (0. lmCi). The distribution of the radioactivity in the liver and remaining carcass, over the 7-day period are reported in Table 1. Radioactivity in the tissues increased slowly with time, reaching a maximum value of 1.8% dose after 24 hr for the liver and 34% dose after 48 hr in the remaining carcass. In the liver, total radioactivity dropped by 60% between 48 hr and 1 week, whereas in the remaining carcass 3H was discharged more slowly with less than 30% being released during the period. The analysis of the labeled compounds in different compartments (Figure l) indicates that after 12 hr the radioactivity was mainly due to unchanged hydrocarbon. However after one week, only 25% of the 3H present in the liver originated from hydrocarbon, whereas approximately 45% was associated with the lipid fraction. In contrast, more than 75% of the total radioactivity in the carcass was a t t r i b u t a b l e to d o d e c y t c y c l o h e x a n e throughout the experiment. The incorporation of radioactivity into hepatic lipids was studied 24 hr after ingestion of 3H-dodecylcyclohexane (Table 2). Phospholipids plus neutral lipids represented more than 90% of the labeling. Specific activity of neutral lipids was approximately twice that of phospholipids and three times higher than that of glycolipids. Analysis of neutral lipid classes (Table 3) shows that most of the radioactivity was in the free fatty acids. Specific activities indicated an heterogeneous distribution in glycerides as in sterols. Separation of the different classes of phospholipids (Table 4) revealed a preferential labeling of glycerophosphatides in comparison with sphingosylphosphatides. Fifty percent of the total 3H was bound to phosphatidyl inositol + phosphatidyl choline. However, when expressed as dpm/mg, the major incorporation of radioactivity was d e t e c t e d in p h o s p h a t i d y l e t h a n o l a m i n e and p h o s p h a t i d y t serine.

Identification of Metabolites

The total lipids from liver sampled at 24 hr were analyzed by TLC followed by radiochromatogram scanning. Comparison with authentic standards chromatographed under identical conditions afforded preliminary identification for most zones. Confirmation of identity of some metabolites was obtained after GC/MS analysis. As indicated in Table 5, TLC of the liver lipids in the solvent system C showed three metabolites from unsaponiliable fraction at Rf 0.98 (I), 0.24 (II) and 0.19 (III) and only one metabolite extracted after acidification of the aqueous phase at Rf 0.40 (IV).

209 Zone 1: This was the principal compound detected in the unsaponifiable fraction. (I) possessed TLC and GC properties identical with those of dodecylcyclohexane. Zone H: This was present in small amounts (6% of the liver radioactivity). The Rf values were similar to those of a long chain alcohol. Acetylation of (II) yielded a radioactive component migrating with the same Rf value as long chain alcohol acetate, suggesting the presence of a hydroxytated metabolite. Solvents C and D clearly separated the compounds hydroxylated on the alkyl chain from cyclohexanol isomers (Table 5). Thus, considering the same Rr value between 4-ethylcyclohexanol and (II), the most likely position :for hydroxylation was on the cyclohexane ring. Analysis of TMS derivative of (II) by GC-MS showed it to be a mixture of two metabolites with retention times of 5.4l (IIa) and 5.8 rain (lib), The mass spectrum of (IIa) showed an apparent M + at m/e 340 indicating a TMS derivative of dodecylcyclohexanol (268 + 73 - 1). Characteristic fragment ions were present at m/e 325 (340 - 15), 129 (CH 2CH-CHO-TMS) which is characteristic of TMS ethers of cyclohexanols, (Niederwieser et aI. 1978), 82 and 83 (cyclohexyl ring), and 73 ((CH3)3-Si). (lib) had qualitatively similar fragmentation patterns, but the relative proportion of the fragment at m/e 129 was 20 for (IIa) compared with tO0 for (IIb). This difference may be due to the position of hydroxylation. The comparison b e t w e e n mass spectra from IIa and IIb and those obtained from TMS derivatives of 2-hydroxycyclohexylacetic acid, 3-hydroxycyclohexylacetic acid and 4-hydroxycyclohexylacetic acid suggest that Illa was 3dodecylcyclohexano] and IIb 4-dodecylcyclohexanol. Zone IlI: This component migrated very close to 2-cyclohexylethanol and represented approximately 8% of the total liver activity. When analyzed by GC-MS, TMS derivative of (III) exhibited a retention_ time of 5.5 rain and a base peak at m/e t 17 (CH3-CHO-TMS). Ot]her significant ions were detected at m/e 325 (34.0 - 15), and 83 (C6 HI~+). Also, none of the signals characteristic of the cyclohexanol fragments was observed. These mass spectral results are consistent with a hydroxyIation occurring on the penultimate carbon of the alkyl chain and leading to cyclohexyldodecane-2-ot. Zone IV: This metabolite accounted for 30% of the liver radioactivity. Based on TLC Rf values of both methylated and underivatized zone IV, this compound was suspected to be a labeled fatty acid. This was confirmed by the GC-MS properties of

210

J-P. Cravedi and J. Tulliez

Table 1. Distribution of radioactivity in liver and carcass of trout, following a single intragastric dose of 3H-dodecylcyclohexane (0.1 mCi) a

Liver Remaining carcass

12 hr

24 hr

48 hr

l week

0.83 • 0.27 17.33 • 5.14

1.79 • 0.31 33.66 • 2.65

1.41 • 0.09 34.12 • 4.72

0.56 • 0.12 24.90 • 3.88

~Values are expressed as percent ofingested dose and represent t h e m e a n s • SDfor 3 animals

2000'

Table 3. Distribution of radioactivity in liver neutral lipids a of trout 24 hours after ingestion of 0.1 mCi of 3H-dodecylcyclohexane

17"21 VA h y d r o c a r b o n

7/ / 7 //

// ~.-

[-]

" -" "//"

1500-

n o n I i pi d s

Percent of total radioactivity

Specific activity (dpm/mg)

//

'~

~

Steryl esters, methyl esters Triglycerides Free fatty acids Cholesterol, diglycerides Monoglycerides

lipids

//1

[000-

~

W'- , ~

//

o ~ //I

//

o~ /11

// // 9 9 0 N

//I

/A //'bO /~ ,g/~" //a e //i u

9.8

24500

16.9 45.0

10500 112500 42000

25,2 3. l

15500

a Neutral lipids free from hydrocarbons b Values represent a single determination from pooled sample from 3 fish

o9 1F-do o 9 oo 9 09

09 a 9 ~ 9 ~ ~o 9 1 4 9

a b L iv er

c

d

a b c Rem

Table 4. Distribution of radioactivity in liver phospholipids of trout 24 hours after ingestion of 0.1 mCi of 3H-dodecylcyclohexane a

d

oining Corcoss

Fig. 1. Evolution and distribution of radioactivity in liver and remaining carcass of trout fed a single dose of 3H-dodecylcyclohexane (0.1 mCi): a = 12 hr, b = 24 hr, c = 48 hr, d = 1 week

Table 2. Distribution of tritium in liver lipids a 24 hours after ingestion of 0.1 mCi of 3H-dodecylcyclohexane

Phospholipids Glycolipids Neutral lipids

Percent of total radioactivity

Specific activity (dpm/mg)

41.9 6.0 52.1

18630 10890 32580

Phosphatidic acid Phosphatidyl ethanolamine Phosphatidyl serine Phosphatidyl choline + Phosphatidyl inositol Sphingomyelin

Percent of total radioactivity

Specific activity (dpm/mg)

11.2 22.3

]4600 29500

8.4

31800

47.5

17900

10.6

13800

Values represent a single determination from pooled sample from 3 fish

a Lipids separated from hydrocarbons b Values represent a single determination from pooled sample from 3 fish

this metabolite which were identical to those of the cyclohexyldodecanoic acid identified in liver following administration of dodecylcyclohexane to rat (Tulliez and Peleran 1977).

Other Metabolites: E f f o r t s t o d e t e c t a m e t a b o l i t e (III') hydroxylated a t t h e e9 p o s i t i o n o f t h e a l k y l c h a i n ( F i g u r e 2) w e r e u n s u c c e s s f u l . Besides, no trace of cyclohexyldecanoic acid or of the inferior homologous acids, corresponding to the first stages of [3-oxydation could be detected.

Naphthenic Hydrocarbons in Trout Liver

21

Table 5. Thin layer chromatographic properties of metabolites of dodecylcyctohexane and authentic reference compounds Rf in solvent a Comp oundd

A

B

C

D

Dodecylcyclohexane I 4-ethylcyclohexanol b II 2-cyclohexyl ethanol b III Cyclohexylacetic acid b IV

0.93 0.92 NT c 0.0 NT 0,0 NT 0.0

0.95 0.95 0.16 0.13 0.10 0.11 NT 0.15

0.99 0.98 0.27 0,24 0.18 0,19 0.44 0.40

0.99 0.98 0,87 0.85 0,73 0,75 0.90 0.90

a The solvents systems were A: hexane; B: hexane/ethyl ether (90:10); C: hexane/etbyl ether/formic acid (80:20:1); D: ethyl ether/ hexane/acetic acid (80:20:1); all proportions v/v b Reference compounds were visualized by I2 vapor o NT = not tested d I, II, 1II, IV = metabolites

~

)Io--CHOH--CH 3

l= (CH2)lI--CH a

i ] I a OH

(CH2)H--CH a

(CH2)I1--CH 3

OH

[ I

l

"ITb

Iff'

(CH2)t~--COOH

9

Fig. 2. M e t a b o l i c p a t h w a y s o f d o d e c y l c y c l o h e x a n e (I) in rainbow trout liver: metabolites I, Iia, IIb, III, and IV were isolated and identified

Discussion

Appreciable amounts of radioactivity were detected in animals as early as 12 hr after dosing. Although the hydrocarbon dose used in this study was 50-fold higher than in an earlier work (Cravedi and Tulliez 1981), radioactivity in the carcass and the liver, expressed as percent of the ingested tritium was similar to the level previously reported, sug-

gesting a residual tissue concentration proportional to the administered dose. Between the 12th hour and 7th day, radioactivity in the carcass remained almost unchanged, in the form of 3H-dodecylcyclohexane. In the liver, 3H was mainly incorporated into the lipid fraction, except during the first hours of the experiment, suggesting that dodecylcyclohexane was biotransformed into lipid components, and/or that 3}t label derived from 3H-dodecylcyclohexane was re-incorporated into tipids. The isolation and identification of labeled cyclohexyldodecanoic acid is in accordance with the former hypothesis, while the radioactivity incorporation into lipids such as free or esterified cholesterol, which had been also noted by Tulliez and Bories (1979) in rats fed 3H-dodecylcyclohexane, lends some support to the latter explanation. The results obtained from the various lipid fractions are comparable in many points to those previously reported with heptadecane (Cravedi and Tulliez, 1986): (I) radioactivity was widespread in the lipid classes of the liver; (2) the recovered 3H resided in equal parts in phosphotipids and in neutral lipids; (3) in neutral lipids, radioactivity was preferentially incorporated into free fatty acids. Besides, the large amounts of phosphatidyl choline plus phosphatidyl inositol present in trout (Castledine and Buckley 1982; Zwingelstein et ai 1978) effectively diluted incoming radioisotope wi~h the result that the specific activity of this fraction was lower than those of phosphatidyl ethanolamine and phosphatidyl serine. However, as reported by Tutliez and Bories (1979) in the rat, the greater deposition of radioactivity in phospholipids occurred in the phosphatidyl choline fraction. Considering the strong influence on membrane

212

fluidity of the composition of its lipids (Hazel 1979) it is possible that incorporation of dodecylcyclohexane metabolites into phospholipids may alter membrane function. Thus, identification of these metabolites was of primary importance. In the related field of alkane metabolism, it has been described in various animal species that the first step of the biotransformations leads to fatty alc o h o l ( K u s u n o s e et al. 1969; M i t c h e l l and H~ibscher 1968; F r o m m e r et al. 1970; PerduDurand and Tulliez 1985). The hydroxylation of dodecylcyclohexane in vivo showed a relatively nonspecific pattern. The main position of hydroxylation was on the alkyl chain (position ~0 and ~o-1). The failure to detect ~o-cyclohexyldodecanol in our samples was probably due to the rapid subsequent oxidation of this alcohol to the corresponding fatty acid. In contrast, the extent of hydroxylation on the ring (position 3 and 4) was approximately 6-fold lower than on the alkyl chain. Moreover, no evidence was obtained for hydroxylation in positions 1 and 2 and this may be due to steric hindrance. This is in agreement with the findings of Elliott et al. (1965); they observed that after methylcyclohexane administration to rabbit, hydroxylation occurred most readily at positions 3 and 4. In the liver of S a l m o g a i r d n e r i fed for nine months a diet containing 1% of d o d e c y l c y c l o hexane, we found substantial amounts of cyclohexyldodecanoic acid (Cravedi and Tulliez 1982b). The present experiment confirms that an important part of the ingested dodecylcyctohexane was retained under this oxidized form in liver lipids. The occurrence of cyclohexyldodecanoic acid among lipids may partly explain the growth depressive effect of dodecylcyclohexane observed by Luquet et al. (1984); such an unusual fatty acid may perturb the membrane organization in which phospholipids are involved. This opinion is fortified by the recent works of Kannenberg et al. (1984), who found in studying the properties of various membranes that the presence of cyclohexyldodecanoic acid in lipids influenced the fluidity and permeability of membranes. Moreover, Sunamoto et al. (1982) showed that inclusion of ~-cyclohexane fatty acids in liposomes decreased the permeability of membranes. Therefore, the general thinking of a complete innocuity of n-alkylcyclohexane hydrocarbons must be questioned. The identification of the various biotransformation products in liver do not exclude the existence of other metabolic routes which may occur in other tissues. However, the results of these experiments show unequivocally that dodecylcyclohexane is rapidly and extensively metabolized by ~o-oxidation

J-E Cravedi and J. Tulliez

and additionally that other minor pathways occur in trout, such as hydroxylation of the subterminal carbon of the alkyi chain or hydroxylation in positions 3 and 4 of the cyclohexane ring. It is not possible from the present experiment to know if the isolated metabolites were subjected to further biotransformation before excretion. Nevertheless, it may be expected that cyclohexyldodecanoic acid could undergo [3-oxidation to cyclohexylacetic acid, as described in rats by Tulliez and Peleran (1977). Thus, the nature of metabolites in excreta have to be examined before a complete scheme of the biotransformation routes of d o d e c y l c y c l o hexane in rainbow trout is proposed. The knowledge of these metabolic processes is essential if in the future we are to have a better understanding of the disposition of these widespread contaminants, naphthenic hydrocarbons, than we do today.

References Castledine AJ, Buckiey JT (1982) Incorporation and turnover of essential fatty acids in phospholipids and neutral lipids of rainbow trout. Comp Biochem Physiol 71B: 119-129 Cravedi JP, Tulliez J (1981) Distribution and elimination routes of a naphthenic hydrocarbon (dodecylcyclohenane) in rainbow trout (Salmo gairdneri). Bull Environ Contam Toxicol 26:337-344 - (1982a) Accumulation, distribution and depuration in trout of naphthenic and isoprenoid hydrocarbons (dodecylcyclohexane and pristane). Bull Environ Contain Toxicol 28:154-161 - (1982b) Chronic ingestion of saturated hydrocarbons by rainbow trout: Influence of dodecylcyclohexane and pristane on lipid metabolism Arch Environ Contam Toxicol 11:719-725 - (1986) Metabolism of n-alkanes and their incorporation into lipids in the rainbow trout. Environ Res (In press) Eltiott TH, Tao RCC, Williams RT (1965) The metabolism of methylcyclohexane. Biochem J 95:70-76 Folch J, Lees H, Sloane-Stanley GH (1957) A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 226:497-509 Frommer U, Ullrich V, Staudinger H (1970) Hydroxylation of aliphatic compounds by liver microsomes.-I. The distribution pattern of isomeric alcohols. Hoppe-Seyler's Z Physiol Chem 351:903-912 Hazel JR (1979) Influence of thermal acclimation on membrane lipid composition of rainbow trout liver. Am J Physiol 236:R91-RI01 Kannenberg E, Blume A, Poralla K (1984) Properties of ~o-cyclohexane fatty acids in membranes. FEBS Lett 172:331334 Korte F, Boedefeld E (1978) Ecotoxicological review of global impact of petroleum industry and its products. Ecotoxicol Environ Safety 2:55-103 Kusunose M, Ichihara K, Kusunose E (1969) Oxidation of nhexadecane by mouse liver microsomal fraction. Biochim Biophys Acta 176:679-681

Naphthenic Hydrocarbons in Trout Liver Lawler GC, Loong WA, Laseter JL (1978) Accumulation of saturated hydrocarbons in tissues of petroleum-exposed mallard ducks (Anas platyrhynchos). Environ Sci Technol 12:47-51 Luqnet P, Cravedi JP, Tulliez J, Bories G (1984) Growth reduction in trout induced by naphthenic and isoprenoid hydrocarbons (dodecylcyclohexane and pristane). Ecotoxicol Environ Safety 8:219-226 Mitchell ME Hiibscher G (1968) Oxidation of n-hexadecane by subcellular preparations of guinea pig small intestine Eur J Biochem 7:90-95 Niederwieser A, Wadman SK, Danks DM (1978) Excretion of cis- and trans-4-hydroxycyclohexylacetic acid in addition to Hawkinsin in a family with a postulated defect of 4-hydroxy-phenylpyruvate dioxygenase. Clin Chim Acta 90:195- 200 Perdu-Durand EF, Tulliez JE (1985) Hydrocarbon hydroxylation system in liver microsomes from four animal species. Fd Chem Toxic 23:363-366 Posthuma J (1977) The composition of petroleum. In AD Mc Intyre and KJ Whittle (eds) Petroleum hydrocarbons in the marine environment. Rapp Pv Rdun Cons int Explor Mer 17l:7-16

213 Sunamoto J, Iwamoto K, Inoue K, Endo T, Nojima S (1982) Liposomal membranes. XI: A suggestion on structural characteristics of acido-thermophilic bacterial membranes. Biochim Biophys Acta 685:283-288 Tulliez JE, Bories GF (1975) M~tabolisme des hydrocarbures paraffiniques et napht~niques chez les animaux sup6rieurs~ II: Accumuiation et mobilisation chez le rat. Ann Nutr Alim 29:2t3-221 (I978) Metabolism of a n-paraffin, heptadecane, in rats. Lipids 13:110-115 (1979) Metabolism of naphthenic hydrocarbons. Utilization of a monocyclic paraffin, dodecylcycMhexane, by rats. Lipids 14:292-297 Tulliez JE, Peleran JC (1977) Glycine conjugation: A metabolic pathway of n-alkyl substituted mono-cycloparaf~ns. FEBS Lett 76:300-302 Zwingelstein G, Abdul Malak N, Brichon G (1978) Effect of environmental temperature on biosynthesis of liver phosphatidylcholine in the trout (Salmo gairdnerii). J Thermal Biology 3:229-233 -

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Manuscript received Jaly 20, 1985 and in revised form October 8, 1985.