Fd Cosmer. Toxicol.
Vol. 14. pp. 577-591.
Pergamon
Press 1976. Printed
in Great Britain
COMPARATIVE METABOLISM OF DIELDRIN IN THE RAT (CFE) AND IN TWO STRAINS OF MOUSE (CFl AND LACG) D. H. HUWN Shell Research
Limited, Tunstall Sittingboume,
Laboratory, Kent ME9
(Received
Sittingbourne 8AG, England
Research
Centre,
18 June 2976)
Abstract-The comparative metabolism of [%]dieldrin has been investigated in control and dieldrinpretreated rats and mice. Metabolites excreted in the urine and faeces, and retained in the livers, kidneys and fat of the five animals in each treatment group were investigated. Pretreated animals received 20 ppm (rats) or 10 ppm (mice) dieldrin in their diet for 28 days prior to the ingestion of [‘4Cjdieldrin. The metabolism of [“C]dieldrin in liver microsomal preparations has also been examined. The major metabolic pathways of dieldrin, leading to 12-hydroxydieldrin, 4,5-trans-dihydroaldrindiol, aldrin-derived dicarboxylic acid and the pentachloroketone are present in both rats and mice. The main differences between the species were a more rapid metabolism of dieldrin in rats, a much greater production of the pentachloroketone by rats, and the production of small amounts of polar urinary metabolites by mice. The two strains of mice were similar to one another in most but not all of the parameters measured. The results are discussed in relation to the increased incidence of liver tumours in CFl mice exposed to dieldrin.
INTRODUCIION
The responses of male CFE rats and male CFl mice to feeding on diets containing dieldrin* are known to differ. Such treatment in both species results in liver enlargement, the reversible proliferation of the smooth endoplasmic reticulum of liver parenchymal cells and increases in liver cytochrome P-450-dependent mono-oxygenase activity (Wright, Potter, Wooder, Donninger & Greenland, 1972), but prolonged feeding of CFl mice on a diet containing 10 ppm clieldrin results, after about 1 yr, in an increased incidence of liver tumours (Walker, Thorpe & Stevenson, 1973), a response which is not observed in CFE rats. The responses in mice are not solely associated with dieldrin. Similar biochemical changes have been observed after the administration of phenobarbitone in the diet to CFl mice (Wright et al. 1972), and pathological responses similar to those seen with dieldrin have been observed with phenobarbitone and DDT (Thorpe & Walker, 1973). Thus, there appears to be an absolute species difference in the response to dieldrin and the other compounds. The LACG strain of mouse appears to be somewhat more resistant than the CFl mouse to the development of liver tumours when given dieldrin for prolonged periods. In males fed diets containing 10 ppm
*The term dieldrin is used throughout this report to mean pure 1,8,9,10,11,11-hexachloro-4,5-exo-epoxy-2,3,7,6endo-2,1,7,8-exo-tetracyclo[6.2.1. 13*6.02~7]dodec-9-ene (von Baeyer/IUPAC nomenclature), trivially known as HEOD, and metabolites will be named according to this numbering. Thus, 12-hydroxydieldrin and 4,5-transdihydroaldrindiol appear throughout this report in place of the older nomenclature for these compounds (9-hydroxydieldrin and 6,7-trans-dihydroaldrindiol). 577
dieldrin for up to 2 yr, the increased incidence barely reached significance (P = @05) compared with that in the controls (E. Thorpe, personal communication, 1976). The biochemical basis of the carcinogenic action of chemical compounds has not yet been established and several approaches are currently being investigated. However, it is now thought that many chemical carcinogens exert their action only after biotransformation to substances possessing electrophilic reactivity or to free-radical intermediates (Magee, 1974; Miller & Miller, 1969; Williams & Rabin, 1971). Of the several types of biotransformation reaction operating within the liver cell (Hutson, 1975), the C- and N-oxygenating enzymes of the liver endoplasmic reticulum (Arrhenius, 1972; Uehleke, 1972) are of major importance in the initial reactions of a lipophilic compound in the sequence that removes most of the compound from the cell. It is these initial reactions, e.g. N-oxygenation of aromatic amines, dealkylation of substituted aromatic amines and nitrosamines and ring oxygenation of polycyclic aromatic hydrocarbons, that also lead to the production of reactive molecular species capable of interacting with’cellular macromolecules (Gelboin, 1969; Sims, 1973; Wang, Rasmussen & Cracker, 1972). Enhanced arylhydrocarbon hydroxylase activity usually provides protection from the carcinogenic effects of polycyclic hydrocarbons and other carcinogens (Gelboin, 1972) because oxygenation is also an important step in detoxication. In addition, other detoxifying enzymes can be induced by the treatments necessary to enhance the oxygenase (Kaplowitz, Kuhlenkamp & Clifton, 1975). However, Gelboin (1972) has emphasized that in certain tissues the reaction mechanisms and sensitivity may be such that the mono-oxygenase produces carcinogenic metabolites.
578
D. H. HUTSON
The reactive metabolitesalso participate in subsequentenzyme-catalysed reactionson membranes or within the cytosol. Thesereactionsmay, or may not, result in detoxication of the reactive species.For example,the conjugationreactions(glucuronidation, sulphationandphosphorylation),hitherto regardedas terminal detoxication reactions,have recently been implicatedin the carcinogenicaction of certain compounds,for examplearomatic N-hydroxyacetamido compounds(Irving, 1972;Lotlikar, 1972). Dieldrin bearslittle structural resemblance to the compoundsdiscussed above, but it clearly interacts with the hepaticmono-oxygenase system.It is a substratefor the enzyme,asshownby Matthews & Matsumura(1969)and in the study now reported, being convertedinto 1Zhydroxydieldrin which is excreted from the liver via the bile as a glucuronide,it inhibits the mono-oxygenation of some other substrates (Wright et al. 1972),and it causesthe induction of this enzymesystem(Wright et al. 1972). In view of the acceptedimportanceof biotransformation in the carcinogenicaction of many compounds,andthe relatively commonoccurrenceof speciesdifferencesin biotransformation,it is important to know whether the absolute speciesdifference betweenthe CFE rat and the LACG and CFl mice in the responseto dieldrin is a consequence of the differencein the metabolismof the compoundin the three types of animal.The formation of a particular metaboliteonly in the CFl mousemight causethe primary lesionleadingto tumour formation. Such a metabolitemight be detectedin the excreta or in a tissueor might be detectableonly as a degradation product (i.e. an excreted metabolite),and if identifiable,its action in other speciescould be assessed. The metabolitesof dieldrin(Fig. 1) in the maleCFl rat havebeeninvestigatedin this laboratory (Baldwin, Robinson& Carrington, 1970; Richardson,Baldwin & Robinson,1968),and the current statusof the sub-
ject has beenreviewed(Bedford & Hutson, 1976).A preliminary study of the comparative excretion of [i4C]dieldrin and its metabolitesin CFE rats and CFl mice has beencarried out (Baldwin, Robinson & Parke, 1972),and the major excreted metabolites of dieldrin in the rat, mouse,rabbit, rhesusmonkey and chimpanzeehave also been compared(Miiller, Nohynek, Woods, Korte & Coulston, 1975).The orientation of the hydroxyl group in the major rat metabolite, IZhydroxydieldrin, has recently been defined,by chemicalstudieson the metabolite(Baldwin, Davis & Thorbum-Burns, 1973) as syn to the epoxide oxygen (Fig. 1). Since there is no evidence for the production of the anti isomer, the isomeric nature of the metabolitewill not be referredto again in this report. The comparativestudy has now been extendedin order to increasethe numberof animals in the experimentalgroups,to includemeasurements of the concentrationsof [14C]dieldrin andits metabolites in tissuessome days after an oral dose of [14Cjdieldrin,to investigatethe effect of pretreatment with dieldrin on the metabolicfate of a singledose of [‘4C]dieldrin, and to comparethe metabolicfates of [14tJdieldrin in CFl and LACG mice. EXPERIMENTAL
Materials. [14CjDieldrin was purchasedfrom the RadiochemicalCentre, Amersham,Bucks, and possessed a specificradioactivity of 79 mCi/mmol.Before useit was purified to 99.9%by thin-layer chromatography (TLC) usinghexane-acetone(95:5, v/v) as solvent. This techniqueremovedabout 3% asimpurities (RF 0, 0.15 and 0.3) from the dieldrin (RF 0.5). The specific radioactivity was verified by scintillation counting and quantitative estimationof the dieldrin by gas-liquidchromatography(GLC). [14ClDieldrin for the mouseexperimentswas dissolvedin dieldrinfree’(c0005 ppm) arachisoil to give a solution of 87.4 &i/ml of oil. The dieldrin to be administered to rats wasdiluted with non-radioactivedieldrin to a specificradioactivity of 25.3 pCi/mg and made up in arachisoil (360 &i/ml). Hexaneand acetonewereredistilledbeforeuse,and other solvents were shown by GLC to contain 200
100 100
35 36
12 13
100
43
14
as representative in each group were combined. Tissues (0%5.Og) were extracted four times by boiling with 25ml hexane-acetone (2: 1, v/v). The extracts were evaporated to low volume, and subsequently reevaporated to low volume with 50ml benzene (later replaced by cyclohexane) to remove water. The residues were then taken up in hexane, the radioactivity was measured and the solution was analysed by GLC for dieldrin, 12-hydroxydieldrin, PCK and photodieldrin (Fig. 1). Column 1 was used for quantitative determination, and column 2 was used to provide additional evidence for the identity of the compounds. 4,5-truns-Dihydroaldrindiol was isolated from the tissue extract (2 ml) by applying the extract to a column containing 3 g 3% deactivated Florisil (Bromshead & Denison Ltd., Welwyn Garden City, Herts.) prewashed with 25 ml hexane-acetone (9:1, v/v). Three fractions were eluted from the column: (1) 25 ml hexane-acetone (99:1, v/v), (2) 25 ml hexane-acetone (9: 1, v/v) and (3) 25 ml hexane-acetone (3:1, v/v). Fraction 3 was boiled to dryness and dissolved in 25 ml acetone. Part of this solution (2 ml) was treated with hexane (2 ml), acetic anhydride (0.5 ml) and 4 drops of boron trifluoride etherate for 10 min. The mixture was diluted to 1Oml with 2% sodium sulphate solution and the upper layer was analysed by GLC for 4,5-truns-dihydroaldrindiol diacetate on column 4. PCK, 12-hydroxydieldrin and dieldrin were measured in the livers of extra groups of CFl and LACG mice and female CFE rats. Weighed whole livers were extracted as described above and the extract was chromatographed as described for 4,5-trans-dihydroaldrindiol. Fraction 1 was analysed for dieldrin (4% GEXE 60 on Diatoport S, 220°C N2), and fraction 2 was analysed for dieldrin, lZhydroxydieldrin and PCK (column 1). Isolation and ident$cation of PCK from mouse carcass. The carcasses of six dieldrin-pretreatedmice of
eachstrain,lessskin,intestinesand liver, wereminced with anhydroussodiumsulphategiving a CFl mince (108.5g) and an LACG mince (128g). The two sampleswereextracted in Soxhletswith hexane-acetone (2:1, v/v). The extracts were boiled to low volume and repeatedly evaporated with redistilled cyclohexaneto removewater. The resultingsolutions werediluted with hexaneand subjectedto a hexanedimethylformamidepartition to remove fat. The extracts were then chromatographedon 3% deactivated Florisil usinghexanecontaining 1.5-10%acetone. The relevant fractionswere combinedto yield 9.76pg PCK from the CFl miceand 14.63pg from the LACG mice.After GLC analysisof the extracts on four different columns(seeTable 7), a further
clean-up on alumina TLC plates (hexane-acetone, 8:2, v/v) was carried out, resulting in 418 pg PCK from the CFl and 3.88 pg from the LACG strains. The sampleswere analysedusing a Finnigan 1015 D massspectrometerGLC combination. Column conditions were 3% OV 225 on 80-100 meshGasChrom Q (2 m x 1.5mm)with a carrier gasflow rate of 20ml helium/mmand a temperatureof 260°C.Under theseconditionsthe retention times of authentic PCK and the two metabolitesampleswere 7.4mir-1. Ions of m/e 192, 261, 289 and 360 were monitored in each caseand the relative intensitiesare shown in Table 1. Preparation
and
extraction
of
liver
microsomes.
Equal weightsof livers from each animal regarded as representativein a treatment group were combined.Liver homogenates (10%)were preparedusing 0.1 M-pOtaSSiUIII phosphatebuffer, pH 7.4.The homogenateswere centrifugedat 9000 g for 20 mm, followed by 100,000g for 60 min to yield a microsomal pellet which was rinsed with phosphatebuffer and re-suspended in the original volumeof buffer. Protein was determined by a modification of the Biuret method(Robinson& Hogden,1940).After determination of radioactivity, l-ml portionsof microsomalsuspensionweremixed with methanol,warmedto boiling point, cooled in ice-water for 10 min and centrifuged. This processwas repeatedtwice using methanol and twice usingethanol.The radioactivity in the supematants was determined, the protein was digested in Protosol and the radioactivity was measured.The first and secondmethanol extracts, containing all of the extracted radioactivity, were combined,allowedto evaporateat room temperature, taken up in hexane,radioassayed,and analysedby GLC usingcolumn 1 followed by column 3. Microsomal mono-oxygenase activity in control and dieldrin-pretreated animals. This enzyme activity was
determinedin microsomalfractions preparedas described above. [14CJChlorfenvinphoswas used as substrateand the procedure was that describedby Donninger,Hutson & Pickering (1972). Microsomal mono-oxygenation of dieldrin in vitro. A male rat which had been fed a diet containing 100ppm phenobarbitonefor 2 wk waskilled by decapitation and liver microsomes werepreparedby standard proceduresfrom a 20%homogenateof the liver in 1.15%KCl. The pellet wasrinsedand then re-suspendedin the samesolutionto give a protein concentration of 10mg/ml.Microsomalincubatesweremade up as follows: [14C]dieldrin (65.4nmolesin 0.2ml acetone,evaporated),microsomalsuspension (2 ml), Tris-HCl buffer (pH 7.65) co-factor solution (1 ml), glucose-6-phosphate dehydrogenase (G6PDI-0, UDPGA (whenadded,4 mg); the final pH was 7.4. The co-factor solutionwascomposedof freshlymixed NADP-glucose-6-phosphatedisodium salt solution (0.5ml) and magnesium chloridesolution(0.5ml). The co-factor concentrationsin the final incubate were: NADP, 0.38mu, MgCl, . 6H20, 4.9mu, glucose-6 phosphate,0.43mu and Tris, 7.4m~. The control reactions were made up as above but omitting G6PDH. In reactionsinvolving the 10,000g supematant of liver homogenate,G6PDH (4ml) was added to the substratewith no additional co-factors. The mixtureswere incubatedat 37°C with ten glassmar-
Metabolism of dieldrin in rats and mice bles of 5 mm diameter. The atmosphere above the solutions was oxygenated every 10min. At 30 and 60 min, 2 ml portions of the solutions were frozen. They were subsequently analysed directly by TLC. Polar metabolites were investigated after precipitation of the protein with 5ml ethanol. [‘4C]Chlorfenvinphos (0.1 111~)was used as a positive control for the measurement of microsomal enzyme activity. This reaction was processed as described previously (Donninger et al. 1972).
581
much.more pronouncedin the two strainsof mouse than in the rat. An exampleof the faecalmetabolites from the three groupsof animalsfollowing administration of a singledose of dieldrin is shownin Fig. 3.
4,5-c&Dihydroaldrindiol in faeces. A recent report (Matthews& McKinney, 1975)hassuggested that formation of the cis-diolmay occur during the metabo-, lismof dieldrin in oioo. This hasbeententatively confirmed by the isolationof a zone of R, 0.31(cf transdiol, RF 0.19) by TLC (solvent A) of an acetone RESULTS extract of day-l rat faeces.This zone was.re-chromaExcretion of Ci4CJdieldrin and its rnetabolites in faeces tographed in solvent B and yielded a radioactive Excretion of radioactivity in faeces. As had pre- metaboliteat R, 0.53.The authentic trans- and cisviously been found in this laboratory, the major isomersmigratedat 0.29and 0.53,respectively,in this excretory route of dieldrin and its metabolitesin both solvent. rats and mice was via the faeces.The mean values Aldrin-derived dicarboxylic acid (ADA) in faeces. for the three animal strains,both dieldrin-pretreated This compoundwas not extractable from faecesin and normal, are shown in Table 2. Excretion was hexane or acetone.Extraction of the pooled faecal more rapid in the rat than in either strain of mouse. residuesfrom normalrats,CFI miceand LACG mice Pretreatmentof CFE rats or CFl micewith dieldrin with methanolafforded69,86 and94%of the remaindid not affect significantlythe excretion of the single ing activity. Of thesevalues,64, 72 and 12%,respectdoseof [14C]dieldrin. Normal LACG mice excreted ively, weretransferableto hexaneafter methyl esterifimuchlessradioactivity than did the other experimen- cation (M. K. Baldwinand D. Bennett,personalcomtal groups.However, after dieldrin pretreatment,the munication,1975).Thesesolutionswerefound to conexcretionwasvery similarto that found with the CFl tain ADA dimethyl ester by GLC analysis using mice. columns1 and 3. The amountsof ADA excretedin Faecal tnetabolites. The major part of the faecal the faeces,expressed as a percentageof the dose of radioactivity in both rats and mice has previously [r4C]dieldrin, were:rat, 1.9; CFl mouse,4.8; LACG beencharacterizedin this laboratory (Baldwin et al. mouse,< @6% 1972)as unchangeddieldrin, syn-12-hydroxydieldrin and 4,5-trans-dihydroaldrindiol(Fig. 1). Thesecom- Excretion of [‘4CJdieldrin and its rnetabolites in urine poundswere shownte be presentin all the faecal Excretion of radioactivity in urine. The meanvalues extracts investigatedin the present experiment by of radioactivity excretedduring days l-8 for the six TLC with authentic standards.Further identification treatmentgroupsare shownin Table 3. Ratsexcreted of dieldrin and 1Zhydroxydieldrin wasmadeby their a significantlyhigher proportion of the radioactivity elution from TLC plates followed by analysis by in the urine than did mice.Excretion of the radioactiGLC. The excretion of radioactive dieldrin, lZhyd- vity by rats wasnot significantlyaltered by pretreatroxydieldrin and 4,5-trans-dihydroaldrindiolas a ment with dieldrin. The values found for mice, function of time after dosing with [14C]dieldrin is although small,may well be too high; the physical shownin Fig. 2. Clear differencesbetweenrats and propertiesof mousefaecesprevent clear separation micecan be seen.The ratio of 12-hydroxy-[r4Cjdiel- from the urine, although this is routinely achieved drin to [14C]dieldrin wasalwaysmuch higher in the in metabolicstudieswith rats. However, the absence quantitiesof dieldrin and 12-hydroxydielrats than in the mice, indicating a more rapid hy- of excessive droxylation reactionand a slightly more rapid excre- drin in the urine samples(as contaminants from tion by the rat. The two strains of mice exhibited faeces)would suggestthat minimumcross-contaminasimilar excretion patterns to one another. Pretreat- tion occurred. Nevertheless,the low volumes and ment of the animals tended to increasethe 12-hy- radioactivity of urine recordeddo not allow distincdroxydieldrin to dieldrin ratio. The effect of pretreat- tion betweenthe two strainsof micenor betweennorment on the excretion of 12-hydroxydieldrin was mal and pretreatedanimals.
Table2. Radioactivity extracted from the faeces of normal and dieldrin-pretreated [“CJdieldrin
Strain CFE Rat
Treatment wow
Normal Pretreated CFl Mouse Normal Pretreated LACG Mouse Normal Pretreated
animals after a single oral dose of
Radioactivityextracted*(% of dose)on day 1
2
3
4
5
6
I
8
11.8 9.1 8.7 19.9 13.1 6.2 11.3 3.3 3.5
6.5 5.7 5.1
4.3
3.9
3.1
2.9
2.7 6.2
1.8 5.3
1.8 2.8 3.8 3.8
10.3 5.0 6.9
58 2.5 5.8
4.3 2.4 4.7
4.0 2.9 5.1
3.4 1.8 4.1
29 25 43
5.8 2.0 4.9
5.7 2.7 4.7
Non-extracted residue(total) 12.1 15.0 9.1 9.3 5.4 7.7
Total 62.4 69.0 51.4 51.5 272 48.8
*Faecalsamples wereextractedovernightwith c. 5vols hexaneand similarlywith acetone.Valuesrepresenttotal daily extractedradioactivity.
582
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, (I d)
30 25
/ t
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./
.’ .I
.I’
/
20-
! ! ! ! I
I’
IO-
./
!
./
!‘ 1
15-
./
./
i $----
i -
,/-
_/-
____----
9)
.’
1234567 Time
.’
./
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I234567 of?eradministration,
days
Fig. 2. The elimination of dieldrin (-), 12-hydroxydieldrin (-.--) and 4,5-fans-dihydroaldrindiol (---) in the faeces of CFE rats (a and d), CFl mice (b and e) and LACG mice (c and f) after a single oral dose of [“‘CJdieldrin to normal (a-c) and dieldrin-pretreated (d-f) animals. Urinary merabolites. Previouswork (Baldwin et al. 1972,and references cited therein)hasshownthat the productsin the urine of malerats after the oral ingestion of [14CJdieldrin include unchanged dieldrin PCK and ADA. Thin-layer chromatographyof urine extractsin severalsolventsfollowed by autoradiography gaveresultsconsistentwith the presenceof these compoundsaccountingfor virtually all of the radioactivity in rat urine (Fig. 4). Of particular interest in the comparativesense,wasthe absencein rat urine
Table 3. Excretion of radioactiuity
of metaboliteswith polarity higher than that of ADA. In mouseurine, however,50%or moreof the urinary radioactivity wascomposedof a complicatedmixture of polar metabolites(RF c. 0.4, solvent C). This is in agreementwith the earlier report of a very polar zone of metabolitedetectedby TLC (Baldwin et al. 1972).A quantitative measureof the polar metabolites (R, 0) relative to ADA (RF 05) and the dieldrin/PCK (RF 0.95)wasobtainedby TLC in solventB, followed by scintillation counting of the three zones. The resultsare shown in Table 4. There would appear to be differencesbetweenthe two strainsof mice in the amount of polar metabolitesproduced.Pretreatmentof rats did not resultin the appearanceof polar metabolites.Pretreatmentof mice did not apparently alter the presenceor the chromatographicprofile of the mixture of polar metabolitesfound in the two strains. Paper chromatography of the mouse-urine extractsrevealedthat 12-hydroxydieldringlucuronide, if present,constituted lessthan 10% of this polar metabolite.Its presencein the urine of both strains of non-pretreatedmousewastentatively confirmedby isolationfrom paperchromatograms(RF 0.1, identical with biosyntheticallypreparedglucu;onide),by TLC of the products(solventC, R, 0.2),and by application of the periodatedegradationtechniqueto the aqueous solutions.The last experimentliberated radioactive materials which partitioned from water to hexane (CFl, 30%; LACG, 23%). A major portion of the radioactivity in the hexane solution was accounted for by GLC as12-hydroxydieldrin(CFI, 63%; LACG, 100%).The overall yield of this metabolite was less than 0.1% of the dosein both strains. Another difference betweenmale CFE rats and male CFl mice reported earlier was the absenceof PCK in the urine of the latter. GLC analysisof mouse urineextracts(normalmice)confirmedthis. However, the urine of dieldrin-pretreatedmicecontaineddetectableamountsof a componentwith the sameretention time as PCK. This evidence,together with the discovery of the compoundin tissuesof pretreatedmice (seebelow),suggested that mouseurine could contain detectablequantitiesof [14C]PCK aswell asthat derived from the non-radioactivedieldrin ingestedduring this pretreatment.Chromatogramsof the mouse urine extracts (TLC, solventsA and B) revealeda trace of radioactive metabolitewith the RF value of
in the urine of normal and dieldrin-pretreated oral dose of [‘4Cjdieldrin
animals
after
a single
Mean urinary radioactivity (% of dose) excreted by groups of fiLe CFE rats Days after dosing
Normal
Pretreated
1 2 3 4 5 6 7 8
1.01 1.03 0.94 0.81 0.78 0.80 0.71 0.48 Total. . . 6.56
CFl mice
LACG mice
Normal
Pretreated
Normal
Pretreated
1.15 0.82 0.66 0.48 0.41 0.32 0.20
0.16 @18 0.28 O-36 0.32 050 050 0.27
0.17 0.12 0.06 0.06 0.09 0.06 0.06 0.09
0.11 0.09 0.13 0.13 0.07 0.06 0.07 0.06
0.08 0.06 DO3 0.05 0.04 0.01 009 0.06
5.48
2.57
0.7 1
0.72
0.42
144
WE .Hexane extract _I_-
rg-
Rat Acetone extract
CFI ’
Hexane extract
Mouse
, LACG Acetone extract
Hexane extract
-
Mouse Acetone extract
Fig. 3. Typical thin-layer chromatograms (solvent A) of extracts of faeces collected on day 4 after dosing non-pretreated animals with [14C]dieldrin.
day 2
days 3-5
days 6-8
PCK
Fig. 4. Thin-layer chromatogram (solvent A) of the metabolites in the urine of rats at various intervals after a single oral dose of [14C]dieldrin.
glucuronyl
I
loooog
dieldrin
Fig. 5. Thin-layer chromatogram (solvent C) of the products of the incubation of [14C]dieldrin with liver micrasomes from a phenobarbitone-treated male rat showing the formation of the glucuronide of 12-hydroxydieldrin.
583
Metabolism of dieldrin in rats and mice Table 4. Relative
quantities of radioactivity lites in the urine of normal
associated with polar metabolites, ADA and neutral animals after a single oral dose of Cf4Cjdieldrin
metabo-
Radioactivity (% extracted from TLC plate) associated with Species CFE rat CFl mouse LACG mouse
Day of urine collection
Polar mouse-urine metabolites (RF 0)
1 2 1 2 1 2
1.1 0.8 48.3 55.9 57.9 88.4
ADA (RF 0.50.7)
-
Dieldrin and PCK (-zzr 0.95)
15.1 17.2 3.2 41 25.3 8.2
83.8 82.0 48.5 40.0 16.8 3.4
ADA = Aldrin-derived dicarboxylic acid The urine was derived from groups of five animals and was analysed by TLC in solvent B. PCK. The compound increased in quantity (reladrin pretreatedanimalswould be expectedto contain tively) with the time after dosing in the pretreated residues derived from non-radioactivedieldrin. animals;the reverseis the casewith the normal aniRadioactivity in the tissues. The radioactivity in the
mals. As is well-known, PCK constitutes about livers,kidneysand fat samplesfrom all groupsof ani25-50% of the urinary metabolitesof [14C]dieldrin malsis shownin Table 6. The valuesare the means in the rat 1 day after dosingand this proportion in- for eachgroup, and excludevaluesderived from anicreaseswith time after dosing.The effect of pretreat- malsthat were clearly atypical. Consideringfirst the ment on [‘4C]PCK excretionby the rat wasnot dra- animalssubjectedto only a singledoseof [‘“Cldielmatic but there was evidencethat the PCK consti- drin, liver and fat residuesof radioactivity werehigher tuted a slightly greater proportion of the urinary in the micethan in the rats. K’idneyresidues, however, metabolitein pretreatedanimalswithin 2 days of the were much higher in the rats, and it is shownbelow treatment (Table 5). The very low quantities of that this differencewasdue to the presenceof PCK [r4C]PCK in the urine of micehaspreventedver-ifica- in the rat kidneys.The two strainsof micehad similar tion of this finding by other methods,but its presence tissueresidues,and the effectsof pretreatmenton the in tissuesshowsthat its formation constitutesa meta- distribution of radioactivity were not marked. Fat bolic route in miceand thereforeits presencein urine residuesappearedto be lower in the pretreatedaniis feasible. mals, but the distinctive rat/mouse difference and CFl/LACG similarity remained. Dieldrin and its metabolites in tissues Identity of metabolites in the tissues. Dieldrin, The animalswerekilled 8 days after receivingthe 12-hydroxydieldrin, PCK, photodieldrin and 4,5singledoseof [’ 4C]dieldrin. Approximately S&70% trans-dihydroaldrindiol were determined by GLC of the radioactivity was excretedduring this period analysisof extractsof pooledtissuesfrom eachgroup leaving residualdieldrin and metabolitesin the tis- of animals.The resultsare shown in Table 6. The sues.Dieldrin is not metabolizedto carbon dioxide meanvaluesfor the total radioactivity in the tissues or to other volatile compoundswhich would be and for the radioactivity extracted from the tissues exhaled(Hedde,Davison & Robbins, 1970).A study are alsoshownin Table 6. Most of the radioactivity of the distribution of the radioactive compoundsis wasextractedexceptin the caseof liver sampleswhen important for the comparisonbetweenthe rat and 28% (1448%) remainedunextracted. The retained mousestrains.In addition, the tissuesfrom the diel- activity was not examined further but it is likely to Table 5. Relative
proportions of [‘VJdieldrin, [‘4CJPCK and Cf4CJADA in the urine dieldrin-pretreated rats after a single oral dose of [‘4CJdieldrin
of normal
and
Radioactivity (% of total metabolites) on day Treatment grow Normal Dieldrin-pretreated
Metabolite
RF
1
2
3-5
6-8
Dieldrin PCK* ADA Dieldrin PCK* ADA
075 050 007 075 050 007
41 42 17 29 51 20
23 58 19 10 71 19
14 73 13 12 69 19
12 78 10 7 82 11
ADA = Aldrin-derived dicarboxylic acid PCK = Pentachloroketone metabolite *GLC analysis for PCK in day-l and day-2 urine samples from both groups of CFl mice and both groups of LACG mice indicated that less than 5% of the urinary radioactivity was composed of PCK. Ethyl acetate extracts of the urine were analysed by TLC in solvent A and the zones were assayed by scintillation counting.
Dieldrinpretreated
Normal
Dieldrinpretreated
Normal
Dieldrinpretreated
Normal
Treatment group
6. Analysis
Liver Kidney Fat Liver Kidney Fat Liver Kidney Fat Liver Kidney Fat Liver Kidney Fat Liver Kidney Fat
Tissue
of the
of groups
0.11 0.65 5.60 0.17 0.61 21.1 0.83 0.23 120J 3.94 1.79 66.0 I.04 0.31 11.1 3.31 1.00 40.7
Dieldrin
tissues
normal
and jive
dieldrin-pretreated
animals
8 days
after
the
Vol. 14. pp. 577-591.
Pergamon
Press 1976. Printed
in Great Britain
COMPARATIVE METABOLISM OF DIELDRIN IN THE RAT (CFE) AND IN TWO STRAINS OF MOUSE (CFl AND LACG) D. H. HUWN Shell Research
Limited, Tunstall Sittingboume,
Laboratory, Kent ME9
(Received
Sittingbourne 8AG, England
Research
Centre,
18 June 2976)
Abstract-The comparative metabolism of [%]dieldrin has been investigated in control and dieldrinpretreated rats and mice. Metabolites excreted in the urine and faeces, and retained in the livers, kidneys and fat of the five animals in each treatment group were investigated. Pretreated animals received 20 ppm (rats) or 10 ppm (mice) dieldrin in their diet for 28 days prior to the ingestion of [‘4Cjdieldrin. The metabolism of [“C]dieldrin in liver microsomal preparations has also been examined. The major metabolic pathways of dieldrin, leading to 12-hydroxydieldrin, 4,5-trans-dihydroaldrindiol, aldrin-derived dicarboxylic acid and the pentachloroketone are present in both rats and mice. The main differences between the species were a more rapid metabolism of dieldrin in rats, a much greater production of the pentachloroketone by rats, and the production of small amounts of polar urinary metabolites by mice. The two strains of mice were similar to one another in most but not all of the parameters measured. The results are discussed in relation to the increased incidence of liver tumours in CFl mice exposed to dieldrin.
INTRODUCIION
The responses of male CFE rats and male CFl mice to feeding on diets containing dieldrin* are known to differ. Such treatment in both species results in liver enlargement, the reversible proliferation of the smooth endoplasmic reticulum of liver parenchymal cells and increases in liver cytochrome P-450-dependent mono-oxygenase activity (Wright, Potter, Wooder, Donninger & Greenland, 1972), but prolonged feeding of CFl mice on a diet containing 10 ppm clieldrin results, after about 1 yr, in an increased incidence of liver tumours (Walker, Thorpe & Stevenson, 1973), a response which is not observed in CFE rats. The responses in mice are not solely associated with dieldrin. Similar biochemical changes have been observed after the administration of phenobarbitone in the diet to CFl mice (Wright et al. 1972), and pathological responses similar to those seen with dieldrin have been observed with phenobarbitone and DDT (Thorpe & Walker, 1973). Thus, there appears to be an absolute species difference in the response to dieldrin and the other compounds. The LACG strain of mouse appears to be somewhat more resistant than the CFl mouse to the development of liver tumours when given dieldrin for prolonged periods. In males fed diets containing 10 ppm
*The term dieldrin is used throughout this report to mean pure 1,8,9,10,11,11-hexachloro-4,5-exo-epoxy-2,3,7,6endo-2,1,7,8-exo-tetracyclo[6.2.1. 13*6.02~7]dodec-9-ene (von Baeyer/IUPAC nomenclature), trivially known as HEOD, and metabolites will be named according to this numbering. Thus, 12-hydroxydieldrin and 4,5-transdihydroaldrindiol appear throughout this report in place of the older nomenclature for these compounds (9-hydroxydieldrin and 6,7-trans-dihydroaldrindiol). 577
dieldrin for up to 2 yr, the increased incidence barely reached significance (P = @05) compared with that in the controls (E. Thorpe, personal communication, 1976). The biochemical basis of the carcinogenic action of chemical compounds has not yet been established and several approaches are currently being investigated. However, it is now thought that many chemical carcinogens exert their action only after biotransformation to substances possessing electrophilic reactivity or to free-radical intermediates (Magee, 1974; Miller & Miller, 1969; Williams & Rabin, 1971). Of the several types of biotransformation reaction operating within the liver cell (Hutson, 1975), the C- and N-oxygenating enzymes of the liver endoplasmic reticulum (Arrhenius, 1972; Uehleke, 1972) are of major importance in the initial reactions of a lipophilic compound in the sequence that removes most of the compound from the cell. It is these initial reactions, e.g. N-oxygenation of aromatic amines, dealkylation of substituted aromatic amines and nitrosamines and ring oxygenation of polycyclic aromatic hydrocarbons, that also lead to the production of reactive molecular species capable of interacting with’cellular macromolecules (Gelboin, 1969; Sims, 1973; Wang, Rasmussen & Cracker, 1972). Enhanced arylhydrocarbon hydroxylase activity usually provides protection from the carcinogenic effects of polycyclic hydrocarbons and other carcinogens (Gelboin, 1972) because oxygenation is also an important step in detoxication. In addition, other detoxifying enzymes can be induced by the treatments necessary to enhance the oxygenase (Kaplowitz, Kuhlenkamp & Clifton, 1975). However, Gelboin (1972) has emphasized that in certain tissues the reaction mechanisms and sensitivity may be such that the mono-oxygenase produces carcinogenic metabolites.
578
D. H. HUTSON
The reactive metabolitesalso participate in subsequentenzyme-catalysed reactionson membranes or within the cytosol. Thesereactionsmay, or may not, result in detoxication of the reactive species.For example,the conjugationreactions(glucuronidation, sulphationandphosphorylation),hitherto regardedas terminal detoxication reactions,have recently been implicatedin the carcinogenicaction of certain compounds,for examplearomatic N-hydroxyacetamido compounds(Irving, 1972;Lotlikar, 1972). Dieldrin bearslittle structural resemblance to the compoundsdiscussed above, but it clearly interacts with the hepaticmono-oxygenase system.It is a substratefor the enzyme,asshownby Matthews & Matsumura(1969)and in the study now reported, being convertedinto 1Zhydroxydieldrin which is excreted from the liver via the bile as a glucuronide,it inhibits the mono-oxygenation of some other substrates (Wright et al. 1972),and it causesthe induction of this enzymesystem(Wright et al. 1972). In view of the acceptedimportanceof biotransformation in the carcinogenicaction of many compounds,andthe relatively commonoccurrenceof speciesdifferencesin biotransformation,it is important to know whether the absolute speciesdifference betweenthe CFE rat and the LACG and CFl mice in the responseto dieldrin is a consequence of the differencein the metabolismof the compoundin the three types of animal.The formation of a particular metaboliteonly in the CFl mousemight causethe primary lesionleadingto tumour formation. Such a metabolitemight be detectedin the excreta or in a tissueor might be detectableonly as a degradation product (i.e. an excreted metabolite),and if identifiable,its action in other speciescould be assessed. The metabolitesof dieldrin(Fig. 1) in the maleCFl rat havebeeninvestigatedin this laboratory (Baldwin, Robinson& Carrington, 1970; Richardson,Baldwin & Robinson,1968),and the current statusof the sub-
ject has beenreviewed(Bedford & Hutson, 1976).A preliminary study of the comparative excretion of [i4C]dieldrin and its metabolitesin CFE rats and CFl mice has beencarried out (Baldwin, Robinson & Parke, 1972),and the major excreted metabolites of dieldrin in the rat, mouse,rabbit, rhesusmonkey and chimpanzeehave also been compared(Miiller, Nohynek, Woods, Korte & Coulston, 1975).The orientation of the hydroxyl group in the major rat metabolite, IZhydroxydieldrin, has recently been defined,by chemicalstudieson the metabolite(Baldwin, Davis & Thorbum-Burns, 1973) as syn to the epoxide oxygen (Fig. 1). Since there is no evidence for the production of the anti isomer, the isomeric nature of the metabolitewill not be referredto again in this report. The comparativestudy has now been extendedin order to increasethe numberof animals in the experimentalgroups,to includemeasurements of the concentrationsof [14C]dieldrin andits metabolites in tissuessome days after an oral dose of [14Cjdieldrin,to investigatethe effect of pretreatment with dieldrin on the metabolicfate of a singledose of [‘4C]dieldrin, and to comparethe metabolicfates of [14tJdieldrin in CFl and LACG mice. EXPERIMENTAL
Materials. [14CjDieldrin was purchasedfrom the RadiochemicalCentre, Amersham,Bucks, and possessed a specificradioactivity of 79 mCi/mmol.Before useit was purified to 99.9%by thin-layer chromatography (TLC) usinghexane-acetone(95:5, v/v) as solvent. This techniqueremovedabout 3% asimpurities (RF 0, 0.15 and 0.3) from the dieldrin (RF 0.5). The specific radioactivity was verified by scintillation counting and quantitative estimationof the dieldrin by gas-liquidchromatography(GLC). [14ClDieldrin for the mouseexperimentswas dissolvedin dieldrinfree’(c0005 ppm) arachisoil to give a solution of 87.4 &i/ml of oil. The dieldrin to be administered to rats wasdiluted with non-radioactivedieldrin to a specificradioactivity of 25.3 pCi/mg and made up in arachisoil (360 &i/ml). Hexaneand acetonewereredistilledbeforeuse,and other solvents were shown by GLC to contain 200
100 100
35 36
12 13
100
43
14
as representative in each group were combined. Tissues (0%5.Og) were extracted four times by boiling with 25ml hexane-acetone (2: 1, v/v). The extracts were evaporated to low volume, and subsequently reevaporated to low volume with 50ml benzene (later replaced by cyclohexane) to remove water. The residues were then taken up in hexane, the radioactivity was measured and the solution was analysed by GLC for dieldrin, 12-hydroxydieldrin, PCK and photodieldrin (Fig. 1). Column 1 was used for quantitative determination, and column 2 was used to provide additional evidence for the identity of the compounds. 4,5-truns-Dihydroaldrindiol was isolated from the tissue extract (2 ml) by applying the extract to a column containing 3 g 3% deactivated Florisil (Bromshead & Denison Ltd., Welwyn Garden City, Herts.) prewashed with 25 ml hexane-acetone (9:1, v/v). Three fractions were eluted from the column: (1) 25 ml hexane-acetone (99:1, v/v), (2) 25 ml hexane-acetone (9: 1, v/v) and (3) 25 ml hexane-acetone (3:1, v/v). Fraction 3 was boiled to dryness and dissolved in 25 ml acetone. Part of this solution (2 ml) was treated with hexane (2 ml), acetic anhydride (0.5 ml) and 4 drops of boron trifluoride etherate for 10 min. The mixture was diluted to 1Oml with 2% sodium sulphate solution and the upper layer was analysed by GLC for 4,5-truns-dihydroaldrindiol diacetate on column 4. PCK, 12-hydroxydieldrin and dieldrin were measured in the livers of extra groups of CFl and LACG mice and female CFE rats. Weighed whole livers were extracted as described above and the extract was chromatographed as described for 4,5-trans-dihydroaldrindiol. Fraction 1 was analysed for dieldrin (4% GEXE 60 on Diatoport S, 220°C N2), and fraction 2 was analysed for dieldrin, lZhydroxydieldrin and PCK (column 1). Isolation and ident$cation of PCK from mouse carcass. The carcasses of six dieldrin-pretreatedmice of
eachstrain,lessskin,intestinesand liver, wereminced with anhydroussodiumsulphategiving a CFl mince (108.5g) and an LACG mince (128g). The two sampleswereextracted in Soxhletswith hexane-acetone (2:1, v/v). The extracts were boiled to low volume and repeatedly evaporated with redistilled cyclohexaneto removewater. The resultingsolutions werediluted with hexaneand subjectedto a hexanedimethylformamidepartition to remove fat. The extracts were then chromatographedon 3% deactivated Florisil usinghexanecontaining 1.5-10%acetone. The relevant fractionswere combinedto yield 9.76pg PCK from the CFl miceand 14.63pg from the LACG mice.After GLC analysisof the extracts on four different columns(seeTable 7), a further
clean-up on alumina TLC plates (hexane-acetone, 8:2, v/v) was carried out, resulting in 418 pg PCK from the CFl and 3.88 pg from the LACG strains. The sampleswere analysedusing a Finnigan 1015 D massspectrometerGLC combination. Column conditions were 3% OV 225 on 80-100 meshGasChrom Q (2 m x 1.5mm)with a carrier gasflow rate of 20ml helium/mmand a temperatureof 260°C.Under theseconditionsthe retention times of authentic PCK and the two metabolitesampleswere 7.4mir-1. Ions of m/e 192, 261, 289 and 360 were monitored in each caseand the relative intensitiesare shown in Table 1. Preparation
and
extraction
of
liver
microsomes.
Equal weightsof livers from each animal regarded as representativein a treatment group were combined.Liver homogenates (10%)were preparedusing 0.1 M-pOtaSSiUIII phosphatebuffer, pH 7.4.The homogenateswere centrifugedat 9000 g for 20 mm, followed by 100,000g for 60 min to yield a microsomal pellet which was rinsed with phosphatebuffer and re-suspended in the original volumeof buffer. Protein was determined by a modification of the Biuret method(Robinson& Hogden,1940).After determination of radioactivity, l-ml portionsof microsomalsuspensionweremixed with methanol,warmedto boiling point, cooled in ice-water for 10 min and centrifuged. This processwas repeatedtwice using methanol and twice usingethanol.The radioactivity in the supematants was determined, the protein was digested in Protosol and the radioactivity was measured.The first and secondmethanol extracts, containing all of the extracted radioactivity, were combined,allowedto evaporateat room temperature, taken up in hexane,radioassayed,and analysedby GLC usingcolumn 1 followed by column 3. Microsomal mono-oxygenase activity in control and dieldrin-pretreated animals. This enzyme activity was
determinedin microsomalfractions preparedas described above. [14CJChlorfenvinphoswas used as substrateand the procedure was that describedby Donninger,Hutson & Pickering (1972). Microsomal mono-oxygenation of dieldrin in vitro. A male rat which had been fed a diet containing 100ppm phenobarbitonefor 2 wk waskilled by decapitation and liver microsomes werepreparedby standard proceduresfrom a 20%homogenateof the liver in 1.15%KCl. The pellet wasrinsedand then re-suspendedin the samesolutionto give a protein concentration of 10mg/ml.Microsomalincubatesweremade up as follows: [14C]dieldrin (65.4nmolesin 0.2ml acetone,evaporated),microsomalsuspension (2 ml), Tris-HCl buffer (pH 7.65) co-factor solution (1 ml), glucose-6-phosphate dehydrogenase (G6PDI-0, UDPGA (whenadded,4 mg); the final pH was 7.4. The co-factor solutionwascomposedof freshlymixed NADP-glucose-6-phosphatedisodium salt solution (0.5ml) and magnesium chloridesolution(0.5ml). The co-factor concentrationsin the final incubate were: NADP, 0.38mu, MgCl, . 6H20, 4.9mu, glucose-6 phosphate,0.43mu and Tris, 7.4m~. The control reactions were made up as above but omitting G6PDH. In reactionsinvolving the 10,000g supematant of liver homogenate,G6PDH (4ml) was added to the substratewith no additional co-factors. The mixtureswere incubatedat 37°C with ten glassmar-
Metabolism of dieldrin in rats and mice bles of 5 mm diameter. The atmosphere above the solutions was oxygenated every 10min. At 30 and 60 min, 2 ml portions of the solutions were frozen. They were subsequently analysed directly by TLC. Polar metabolites were investigated after precipitation of the protein with 5ml ethanol. [‘4C]Chlorfenvinphos (0.1 111~)was used as a positive control for the measurement of microsomal enzyme activity. This reaction was processed as described previously (Donninger et al. 1972).
581
much.more pronouncedin the two strainsof mouse than in the rat. An exampleof the faecalmetabolites from the three groupsof animalsfollowing administration of a singledose of dieldrin is shownin Fig. 3.
4,5-c&Dihydroaldrindiol in faeces. A recent report (Matthews& McKinney, 1975)hassuggested that formation of the cis-diolmay occur during the metabo-, lismof dieldrin in oioo. This hasbeententatively confirmed by the isolationof a zone of R, 0.31(cf transdiol, RF 0.19) by TLC (solvent A) of an acetone RESULTS extract of day-l rat faeces.This zone was.re-chromaExcretion of Ci4CJdieldrin and its rnetabolites in faeces tographed in solvent B and yielded a radioactive Excretion of radioactivity in faeces. As had pre- metaboliteat R, 0.53.The authentic trans- and cisviously been found in this laboratory, the major isomersmigratedat 0.29and 0.53,respectively,in this excretory route of dieldrin and its metabolitesin both solvent. rats and mice was via the faeces.The mean values Aldrin-derived dicarboxylic acid (ADA) in faeces. for the three animal strains,both dieldrin-pretreated This compoundwas not extractable from faecesin and normal, are shown in Table 2. Excretion was hexane or acetone.Extraction of the pooled faecal more rapid in the rat than in either strain of mouse. residuesfrom normalrats,CFI miceand LACG mice Pretreatmentof CFE rats or CFl micewith dieldrin with methanolafforded69,86 and94%of the remaindid not affect significantlythe excretion of the single ing activity. Of thesevalues,64, 72 and 12%,respectdoseof [14C]dieldrin. Normal LACG mice excreted ively, weretransferableto hexaneafter methyl esterifimuchlessradioactivity than did the other experimen- cation (M. K. Baldwinand D. Bennett,personalcomtal groups.However, after dieldrin pretreatment,the munication,1975).Thesesolutionswerefound to conexcretionwasvery similarto that found with the CFl tain ADA dimethyl ester by GLC analysis using mice. columns1 and 3. The amountsof ADA excretedin Faecal tnetabolites. The major part of the faecal the faeces,expressed as a percentageof the dose of radioactivity in both rats and mice has previously [r4C]dieldrin, were:rat, 1.9; CFl mouse,4.8; LACG beencharacterizedin this laboratory (Baldwin et al. mouse,< @6% 1972)as unchangeddieldrin, syn-12-hydroxydieldrin and 4,5-trans-dihydroaldrindiol(Fig. 1). Thesecom- Excretion of [‘4CJdieldrin and its rnetabolites in urine poundswere shownte be presentin all the faecal Excretion of radioactivity in urine. The meanvalues extracts investigatedin the present experiment by of radioactivity excretedduring days l-8 for the six TLC with authentic standards.Further identification treatmentgroupsare shownin Table 3. Ratsexcreted of dieldrin and 1Zhydroxydieldrin wasmadeby their a significantlyhigher proportion of the radioactivity elution from TLC plates followed by analysis by in the urine than did mice.Excretion of the radioactiGLC. The excretion of radioactive dieldrin, lZhyd- vity by rats wasnot significantlyaltered by pretreatroxydieldrin and 4,5-trans-dihydroaldrindiolas a ment with dieldrin. The values found for mice, function of time after dosing with [14C]dieldrin is although small,may well be too high; the physical shownin Fig. 2. Clear differencesbetweenrats and propertiesof mousefaecesprevent clear separation micecan be seen.The ratio of 12-hydroxy-[r4Cjdiel- from the urine, although this is routinely achieved drin to [14C]dieldrin wasalwaysmuch higher in the in metabolicstudieswith rats. However, the absence quantitiesof dieldrin and 12-hydroxydielrats than in the mice, indicating a more rapid hy- of excessive droxylation reactionand a slightly more rapid excre- drin in the urine samples(as contaminants from tion by the rat. The two strains of mice exhibited faeces)would suggestthat minimumcross-contaminasimilar excretion patterns to one another. Pretreat- tion occurred. Nevertheless,the low volumes and ment of the animals tended to increasethe 12-hy- radioactivity of urine recordeddo not allow distincdroxydieldrin to dieldrin ratio. The effect of pretreat- tion betweenthe two strainsof micenor betweennorment on the excretion of 12-hydroxydieldrin was mal and pretreatedanimals.
Table2. Radioactivity extracted from the faeces of normal and dieldrin-pretreated [“CJdieldrin
Strain CFE Rat
Treatment wow
Normal Pretreated CFl Mouse Normal Pretreated LACG Mouse Normal Pretreated
animals after a single oral dose of
Radioactivityextracted*(% of dose)on day 1
2
3
4
5
6
I
8
11.8 9.1 8.7 19.9 13.1 6.2 11.3 3.3 3.5
6.5 5.7 5.1
4.3
3.9
3.1
2.9
2.7 6.2
1.8 5.3
1.8 2.8 3.8 3.8
10.3 5.0 6.9
58 2.5 5.8
4.3 2.4 4.7
4.0 2.9 5.1
3.4 1.8 4.1
29 25 43
5.8 2.0 4.9
5.7 2.7 4.7
Non-extracted residue(total) 12.1 15.0 9.1 9.3 5.4 7.7
Total 62.4 69.0 51.4 51.5 272 48.8
*Faecalsamples wereextractedovernightwith c. 5vols hexaneand similarlywith acetone.Valuesrepresenttotal daily extractedradioactivity.
582
D. H. HUIXIN LO)
, (I d)
30 25
/ t
./
./
.’ .I
.I’
/
20-
! ! ! ! I
I’
IO-
./
!
./
!‘ 1
15-
./
./
i $----
i -
,/-
_/-
____----
9)
.’
1234567 Time
.’
./
./
I234567 of?eradministration,
days
Fig. 2. The elimination of dieldrin (-), 12-hydroxydieldrin (-.--) and 4,5-fans-dihydroaldrindiol (---) in the faeces of CFE rats (a and d), CFl mice (b and e) and LACG mice (c and f) after a single oral dose of [“‘CJdieldrin to normal (a-c) and dieldrin-pretreated (d-f) animals. Urinary merabolites. Previouswork (Baldwin et al. 1972,and references cited therein)hasshownthat the productsin the urine of malerats after the oral ingestion of [14CJdieldrin include unchanged dieldrin PCK and ADA. Thin-layer chromatographyof urine extractsin severalsolventsfollowed by autoradiography gaveresultsconsistentwith the presenceof these compoundsaccountingfor virtually all of the radioactivity in rat urine (Fig. 4). Of particular interest in the comparativesense,wasthe absencein rat urine
Table 3. Excretion of radioactiuity
of metaboliteswith polarity higher than that of ADA. In mouseurine, however,50%or moreof the urinary radioactivity wascomposedof a complicatedmixture of polar metabolites(RF c. 0.4, solvent C). This is in agreementwith the earlier report of a very polar zone of metabolitedetectedby TLC (Baldwin et al. 1972).A quantitative measureof the polar metabolites (R, 0) relative to ADA (RF 05) and the dieldrin/PCK (RF 0.95)wasobtainedby TLC in solventB, followed by scintillation counting of the three zones. The resultsare shown in Table 4. There would appear to be differencesbetweenthe two strainsof mice in the amount of polar metabolitesproduced.Pretreatmentof rats did not resultin the appearanceof polar metabolites.Pretreatmentof mice did not apparently alter the presenceor the chromatographicprofile of the mixture of polar metabolitesfound in the two strains. Paper chromatography of the mouse-urine extractsrevealedthat 12-hydroxydieldringlucuronide, if present,constituted lessthan 10% of this polar metabolite.Its presencein the urine of both strains of non-pretreatedmousewastentatively confirmedby isolationfrom paperchromatograms(RF 0.1, identical with biosyntheticallypreparedglucu;onide),by TLC of the products(solventC, R, 0.2),and by application of the periodatedegradationtechniqueto the aqueous solutions.The last experimentliberated radioactive materials which partitioned from water to hexane (CFl, 30%; LACG, 23%). A major portion of the radioactivity in the hexane solution was accounted for by GLC as12-hydroxydieldrin(CFI, 63%; LACG, 100%).The overall yield of this metabolite was less than 0.1% of the dosein both strains. Another difference betweenmale CFE rats and male CFl mice reported earlier was the absenceof PCK in the urine of the latter. GLC analysisof mouse urineextracts(normalmice)confirmedthis. However, the urine of dieldrin-pretreatedmicecontaineddetectableamountsof a componentwith the sameretention time as PCK. This evidence,together with the discovery of the compoundin tissuesof pretreatedmice (seebelow),suggested that mouseurine could contain detectablequantitiesof [14C]PCK aswell asthat derived from the non-radioactivedieldrin ingestedduring this pretreatment.Chromatogramsof the mouse urine extracts (TLC, solventsA and B) revealeda trace of radioactive metabolitewith the RF value of
in the urine of normal and dieldrin-pretreated oral dose of [‘4Cjdieldrin
animals
after
a single
Mean urinary radioactivity (% of dose) excreted by groups of fiLe CFE rats Days after dosing
Normal
Pretreated
1 2 3 4 5 6 7 8
1.01 1.03 0.94 0.81 0.78 0.80 0.71 0.48 Total. . . 6.56
CFl mice
LACG mice
Normal
Pretreated
Normal
Pretreated
1.15 0.82 0.66 0.48 0.41 0.32 0.20
0.16 @18 0.28 O-36 0.32 050 050 0.27
0.17 0.12 0.06 0.06 0.09 0.06 0.06 0.09
0.11 0.09 0.13 0.13 0.07 0.06 0.07 0.06
0.08 0.06 DO3 0.05 0.04 0.01 009 0.06
5.48
2.57
0.7 1
0.72
0.42
144
WE .Hexane extract _I_-
rg-
Rat Acetone extract
CFI ’
Hexane extract
Mouse
, LACG Acetone extract
Hexane extract
-
Mouse Acetone extract
Fig. 3. Typical thin-layer chromatograms (solvent A) of extracts of faeces collected on day 4 after dosing non-pretreated animals with [14C]dieldrin.
day 2
days 3-5
days 6-8
PCK
Fig. 4. Thin-layer chromatogram (solvent A) of the metabolites in the urine of rats at various intervals after a single oral dose of [14C]dieldrin.
glucuronyl
I
loooog
dieldrin
Fig. 5. Thin-layer chromatogram (solvent C) of the products of the incubation of [14C]dieldrin with liver micrasomes from a phenobarbitone-treated male rat showing the formation of the glucuronide of 12-hydroxydieldrin.
583
Metabolism of dieldrin in rats and mice Table 4. Relative
quantities of radioactivity lites in the urine of normal
associated with polar metabolites, ADA and neutral animals after a single oral dose of Cf4Cjdieldrin
metabo-
Radioactivity (% extracted from TLC plate) associated with Species CFE rat CFl mouse LACG mouse
Day of urine collection
Polar mouse-urine metabolites (RF 0)
1 2 1 2 1 2
1.1 0.8 48.3 55.9 57.9 88.4
ADA (RF 0.50.7)
-
Dieldrin and PCK (-zzr 0.95)
15.1 17.2 3.2 41 25.3 8.2
83.8 82.0 48.5 40.0 16.8 3.4
ADA = Aldrin-derived dicarboxylic acid The urine was derived from groups of five animals and was analysed by TLC in solvent B. PCK. The compound increased in quantity (reladrin pretreatedanimalswould be expectedto contain tively) with the time after dosing in the pretreated residues derived from non-radioactivedieldrin. animals;the reverseis the casewith the normal aniRadioactivity in the tissues. The radioactivity in the
mals. As is well-known, PCK constitutes about livers,kidneysand fat samplesfrom all groupsof ani25-50% of the urinary metabolitesof [14C]dieldrin malsis shownin Table 6. The valuesare the means in the rat 1 day after dosingand this proportion in- for eachgroup, and excludevaluesderived from anicreaseswith time after dosing.The effect of pretreat- malsthat were clearly atypical. Consideringfirst the ment on [‘4C]PCK excretionby the rat wasnot dra- animalssubjectedto only a singledoseof [‘“Cldielmatic but there was evidencethat the PCK consti- drin, liver and fat residuesof radioactivity werehigher tuted a slightly greater proportion of the urinary in the micethan in the rats. K’idneyresidues, however, metabolitein pretreatedanimalswithin 2 days of the were much higher in the rats, and it is shownbelow treatment (Table 5). The very low quantities of that this differencewasdue to the presenceof PCK [r4C]PCK in the urine of micehaspreventedver-ifica- in the rat kidneys.The two strainsof micehad similar tion of this finding by other methods,but its presence tissueresidues,and the effectsof pretreatmenton the in tissuesshowsthat its formation constitutesa meta- distribution of radioactivity were not marked. Fat bolic route in miceand thereforeits presencein urine residuesappearedto be lower in the pretreatedaniis feasible. mals, but the distinctive rat/mouse difference and CFl/LACG similarity remained. Dieldrin and its metabolites in tissues Identity of metabolites in the tissues. Dieldrin, The animalswerekilled 8 days after receivingthe 12-hydroxydieldrin, PCK, photodieldrin and 4,5singledoseof [’ 4C]dieldrin. Approximately S&70% trans-dihydroaldrindiol were determined by GLC of the radioactivity was excretedduring this period analysisof extractsof pooledtissuesfrom eachgroup leaving residualdieldrin and metabolitesin the tis- of animals.The resultsare shown in Table 6. The sues.Dieldrin is not metabolizedto carbon dioxide meanvaluesfor the total radioactivity in the tissues or to other volatile compoundswhich would be and for the radioactivity extracted from the tissues exhaled(Hedde,Davison & Robbins, 1970).A study are alsoshownin Table 6. Most of the radioactivity of the distribution of the radioactive compoundsis wasextractedexceptin the caseof liver sampleswhen important for the comparisonbetweenthe rat and 28% (1448%) remainedunextracted. The retained mousestrains.In addition, the tissuesfrom the diel- activity was not examined further but it is likely to Table 5. Relative
proportions of [‘VJdieldrin, [‘4CJPCK and Cf4CJADA in the urine dieldrin-pretreated rats after a single oral dose of [‘4CJdieldrin
of normal
and
Radioactivity (% of total metabolites) on day Treatment grow Normal Dieldrin-pretreated
Metabolite
RF
1
2
3-5
6-8
Dieldrin PCK* ADA Dieldrin PCK* ADA
075 050 007 075 050 007
41 42 17 29 51 20
23 58 19 10 71 19
14 73 13 12 69 19
12 78 10 7 82 11
ADA = Aldrin-derived dicarboxylic acid PCK = Pentachloroketone metabolite *GLC analysis for PCK in day-l and day-2 urine samples from both groups of CFl mice and both groups of LACG mice indicated that less than 5% of the urinary radioactivity was composed of PCK. Ethyl acetate extracts of the urine were analysed by TLC in solvent A and the zones were assayed by scintillation counting.
Dieldrinpretreated
Normal
Dieldrinpretreated
Normal
Dieldrinpretreated
Normal
Treatment group
6. Analysis
Liver Kidney Fat Liver Kidney Fat Liver Kidney Fat Liver Kidney Fat Liver Kidney Fat Liver Kidney Fat
Tissue
of the
of groups
0.11 0.65 5.60 0.17 0.61 21.1 0.83 0.23 120J 3.94 1.79 66.0 I.04 0.31 11.1 3.31 1.00 40.7
Dieldrin
tissues
normal
and jive
dieldrin-pretreated
animals
8 days
after
the
