Chem.-Bioi. Intemctions. 46 (1983) 39-M Elaevier Scientific Publishers Ireland Ltd.

39

COMPARISON OF CYTOCHROME P-4.50-DEPENDENT TABOLIS IN DIFFERENT DEVELOPMENTAL STAGES OF ~~0~0~~~ ~~OGA~~E~

I. HiiLLST6M. A. BLANCK’ and S. ATUMAb

Department of %rieok?gicai Genetics. W&?&erg Labnmtory. University of Stxkholm. I0691 Stockholm, ‘Department of Medical Nutrition, Huddinge LJnivPrsity Hospital. Iz 69, S-141 86 Huddinge and bInslttute for Chemical Environmental Analyses, Wailenbeg Laboratory, ITni- versity of Stockholm, 106 91 Stoekhulm (Sweden)

(Received November 15th. 1982) (Revision received February 1 lth, 1983) (Accepted February 16th. 1983)

SUMMARY

The activities of -everal drug metaboiizing enzymes were compared in microsomes from larvae and adult Drosophila. The cytochrome P-450 content and the benzo[alpyrene (BP) hydroxylation, p-niiroanisole demethylation and 3- and 4-hydroxylation of biphenyl were 4-20-fold higher in microsomes from adult flies, while 7.ethoxycoumarin deethyfase activity and cytochromr c reductase activity were about the same in the two stages. 2-OH-biphenyl was formed in trace amounts by microsomes from adult flies but not to any detectable amount by microsomes from Iarvae. Pretreatment with pheno- barbital (PB), Aroclor 1254 (PCB) or i;l-naphthoflavone (BNF) increased the cytochrome P-450 content and the various cytochrome P-450-mediated reac- tions up to ‘I-fold in larvae. The effects of the pretreatmenzs were weaker in adult flies, where the increase never was more than 3-fold. and man!: reactions were unaffected by the pretreatment% BNF was thus inefficient in enhancing all reactions, except a slight (1.3-fold) increase in the formation of 4-OH-biphenyl. Microsomes from both stages exhibited increases in specific protein bands with apparent molecular weights of 51000-58 000 in the sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis following treatment with PB, PCB and BNF. Differences were observed brtwrrn larvae and adults with respect rJoth to the number of and the molecular weif:hts af the increased protein bands.

A~~~viat.iol~s: BNF, ~.nnphthoflavone~ BP, benzola ipyrenr: BSA. bovine serum ttlbumtn; CBR. C lomassie Brilliant Blue: PAH, polycyclic aromatic hydrocarbons: PB. phenobarbital: PC& palychlorinatwl biphenyls (Aroclor 1264): SDS, sodium dodecyl sulphate: TCDD. tetrachloro- &benzo-pdioxin: TMBZ, 3,3’,6,5’-tetramethylbenzidine.

0009.2797/88/$03.00 $3 1963 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

40

Key worcls: Drosoplhilu - Cytochrome P-450 -Metabolism

INTRODUCTION

Xcnobiotic metabolism, as performed by the cytochrome P-450 enzyme system, has been shown to vary both quantitatively and qualitatively during pre- and postnatal development [l-3]. Consequently, the formation of toxic and carcinogenic mietabolites and thus the sensitivity to detrimental effects of environmental exposure could be quite different at different developmental stages

Drosophila melanogaster, one of the most important test organisms in the detection of genotoxic substances,, was shown to respond differently to mutagen treatment as a larvae compared to the adult fly [4,5]. Nevertheless, both stages are used as experimental organisms in mutation tests.

The occurrence ol cytochrome P-450 and xenobiotic metabolism have been shown both in larvae and in adult flies 1691. The aim of this study was to present a more det.ailed investigation of the metabolic capacity of the two stages. The basal questions to answer concerned the capacity of subcellular fractions from both1 stages to perform chosen enzymatic reactions, as well as to detect differences in the sensitivity to enzyme-inducing agents and varia- tions in the pattern of microsomal. proteins in the SDS-polyacrylamide gel electrophoresis. An increased knowledge of these parameters might con= tribute to the ablility to evaluate mutagenicity assays with Drosophila melanogaster as te,at organism.

MATERIALS AND METHODS

Chemicals BP was obtained from Sigma Chemical Co (St. Louis, MO), PCB (Aroclor

1254) from Monsanto Chemical Co (St. Louis, MO) and BNF from Aldrich Chemical Co Inc (Milwaukee, WI). Pentafhtorobenzoylchloride was purchased from Ega Chemie (:Steinheim/Albuch, F.R.G.). Other chemicals obtained from local commercial suppliers were of analytical grade.

Animals and pretreatments In the present study larvae and adult flies of strain Karsnlis 60 were used.

To prevent an intrastrain variation, an isogenic stock obtained through long-time brother-sister crosses was used. The strain also carries the markers w(X) and lBS(Y). This strain was kindly provided by Dr. K. G. Liining, Genetic Institute, University of Stockholm, Below this strain is referred to as Karsnas 60,.

Larva,e (60 -+ 12-h-old) were pretreated with PB (2 mg/mll dissolved in

41

Bacto-Penassay Broth (antibiotic medium 3), PCB (2mglml) or BNF (1 mg/ml) diesolved in Tween 80 and emulsified in the same medium (5% v/v). The larvae were pretreated for 24 h as previously described [s].

Adult flies (2-5 days post eclosion) were exposed to the pretreatment substances dissolved in water G’B) or corn oil (PCB, BNF) and mixed in the ordinary corn-agar substrate. The flies were pretreated for 18 h with 1 mg PBlml or 0.5 mg PCB/ml. For BNF, the highest dose studied, 5 nag/ml, was used for further metabolic studies. The flies were kept on empty vials for 4 h prior to pretreatments.

Isolation of microsomes Microsomes were prepared, as previously described for larvae IS], by

homogenization of larvae or adult flies in ice-cold 0.2 M phosphate buffer (pH 7.5) (0.25g larvae/ml and 0.1-0.2g flies/ml) in a glass-Teflon Potter-Elveh- jem homogenizer. The larvae homogenate was centrifuged for 600 Y g for 2 min, followed by 15 000 x g for 10 min. The supernatant was layered over 0.3M sucrose in the phosphate buffer according to the method of Arrhenius [lo] and centrifuged at 115 000 x g for 60 min. Microsomes from adult flies were obtained with the same technique, but the 600 * g centrifugation W~IS omitted, and the 15 000 x g supernatant was filtered through nylon cloth prior to the ultra centrifugation. The microsomal pellets were resuspended in buffer and used for enzymatic assays within 1 h, or rapidly frozen and stored at -7O”C, a procedure that caused a negligible decresse in enzyme activit) for a storage time of up to 2-3 months. For the heme staining (see section for SDS gel electrophoresis) the use of freshly prepared microsomes was however absolutely necessary. During preparation and analysis of the microsomes. the temperature was kept at 2-4”C.

Assays The reduced, CO-bound cytochrome P-450 difference spectra were recorded

according to Omura and Sato 1111, NADPH-cytochrome c reductase was measured by the method described by Maze1 [ 121 and protein was determined according to Lowry with bovine serum albumin as the standard [131. BP monooxygenase activity was measured by the fluorimetric method of Dehnen et al. [la]. Incubation conditions were as previously described [S]. For the determination of the demethylation of p-nitroanisole the incubation mixture contained l-3 mg microsomal protein, 4 pmol sodium isocit (*ate. 0.8 c~mol NADP’. 4 pmol MgCl,, 40 pmol Tris-HCl buffer (pH 7.8) and isocitratr dehydrogenase with a reducing capacity of 1.24 pmol NADP.imin in a final volume of 2ml. After preincubation for 2 min at 37°C. the reaction was started by the addition of 20 ~1 1 M PNA in acetone, incubated for 10 min and stopped with 0.5 ml ice-cold 10% (v/v) acetic acid. T> decrease back- ground absorption the sample was extracted with ethyl acetatelhexane (1: 1, v/v, 2.5 ml) and the organic phase was reextracted with 1 ml 1 M NaJJO,I before quantitation of the p-nitrophenol formed as measured by the ab-

42

sorption at 4OOnm (according to Dr. D. Hultmark, pers. comm.). The 00 deethylation of 7-ethoxycoumarin WI&S determined directly in the fluorescence cuvette at 37°C according to Ullrich and Weber [151 with 0.25-0.5 mg micro- somal protein, 2 mg bovine serum albumin (BSA), 100 mm01 Tris-HCI buffer (pH 7.6) and 50 pmol ‘I-ethoxycoumarin in a l-ml incubation. The reaction was started by the addition of 100 nmol NADPH. For the determination of biphenyl hydroxylaltion 0.2-0.5 mg microsomal protein, 1.8 pmol NADPH, 1.4 pmol NADH, 4.!5 pmol MgClp and 1 mg BSA in a total volume of 1.25 ml Tris-HCl buffer (O.O5M, pH 8.5), were preincubated for 2min, started with 0.3 pmol biphenyl in 25 pl acetone and incubated for 10 min at 37°C. The reaction was stopped with the addition of 0.5 ml 2 M HCl. The analysis was performed as a modification of the method of Rehnberg [161, consisting of extraction with hexane (2 ml), reextraction with 0.5M NaOH (2ml), and benzoylation of the phenolic metaballites formed with 25 ~1 freshly made 10% pentafluorobenzoylchloride in toluene after the addition of 1 ml hexane and 3 ml 1 M NaH CO, to the aqueous NaOH-phase. The organic phase was shaken with a fresh 2-ml portion of 0.5 M NaOH for 30 min and recentrifuged prior to the quantitation. The biphenyl metabolites were quantitated on a Varian Model 3700 gas chromatograph with aNi electron capture detector; 25 M quartz glass capillary (i.d. 0.32 mm) coated with SE 54 (l%vinyl, 5%phenyl methyl silicone). For the fluorescence and absorbance measurements a Shimadzu RF-5102C and uV-500, respectively, were used.

SDS-polyacrylamisle gel electrophoresis SDS-polyacrylamide gel electrophoresis was performed according to the

method described by Laemmli [17]. A total amount of 25 pg microsomal protein was applied to each well at the gel. The stacking gel contained 4.5% polyacrylamide and the separating gel 9%. After staining the gels for protein with Coomassie Brilliant Blue (CBB), densitometric tracing was performed at 550 nm.

For the identification of heme-co’ntaining protein bands that might consist of cytochrome P-450 species, separate gels were also run and stained for peroxidase activity with 3,3’,5,5’-t.etramethylbenzidine (TMBZ). To get ap- propriate detection sensitivity for the heme staining, a total amount of 500 pg microsomal protein had to be applied to each well. The method described by Sinclair et al. [18] was used with the following modifications: electrophoresis was run on 2mm thick gels; the polyacrylamide concen= trations in the stacking and th’e separating gels were 4.5% and 9.08, respectively, as described above; Ehomophenol Blue (0.002% w/v) was added to the samples for the visualization of front movement during the electro- phoresis. The densitometric tracing of the gels were performed at 600 nm. Identific,ation of the molecular weight regions where the protein bands staining for heme were located was made using catalase and liver micro= somes from a BIG-treated rat as reference proteins. Whereas frozen material could be used for the gels stained for protein without any change in the electrophoret.ic pattern, only freshly prepared microsomes could be used when gels were to be stained from heme.

43

RESULTS

The microeomee from adult flies turned out to be more stable and easier to prepare and handle than the larval fraction (the stability and properties of the latter were described in a previous paper [81). This is logical in view of the enormous food consumption during the larval stages, increasing the amount of digeetive and proteolytic enzymes present in the preparations. The adult microsomes could be used for enzyme assays for at least two hours without substantial loss of activity, provided the temperature was kept well below 4°C. To ensure a correct comparison between the stages all measure- ments were, however, made within 0.5-l h, during which time the larval preparations did not decline in activity.

The induction performance of larvae were as previously described [S]. For adult flies, the optimal concentrations of PB and PCB at a 24-h pretreatment are shown in Fig. 1. For BNF, no significant increase in cytochrome P-450 content (Fig. 1) or BP monooxygenase activity (data not shown) was observed in the dose range 0.05-5 mg/ml; the highest dose was chosen for the metabol- ism studies. The time-dependence for the PB-induction at the selected dose 1 mg/ml can be seen in Fig. 2. The optimal pretreatment time was found to be 18 h; keeping the flies on empty vials for 4 h prior to the treatment slightly increased the effect of the pretreatments.

The effect of the various pretreatments on cytochrome P-450 content and NADPH-cytochrome c reductase activity are shown in Table I. The cq’to-

chrome P-450 content was nearly I-fold higher in adult flies when compared

PB

PC 6

BNF

0,Ol 0.05 0.1 0,5 1 2 2.5 5 10 dose. mg ,,,,

Fig. 1. The relation between pretreatment dose and cytochrome P-450 content in adult flies of the Kawniia 60~ strain. The pretreatment substances were administrated in the food for 24 h.

I I 0 6 12 18 24 36 j2 Wetreatment ~trne. hours

Fig. 2. PB induction of cytochrome P-450 as a function of pretreatment time in adult Karsniis 60~ flies. The pretreatment dose was 1 mg PB/ml.

to larvae. The relative increase after PB and PCB pretreatment was, howev- er, more marked in larvae. A 3-4.fold increase in larvae as a response to PB or PCB treatment should be compared to a 1.9.fold and 1.7.fold increase, respectively in adu.lts. The absorption maximum of the reduced P-450X0 complex was 450 nm in both untreated larvae and adult flies. In larvae, PB treatment caused a 2nm red shift and PCB a 2nm blue shift in the absorption maximum, while it was not changed in adults after the various pretreatments. NADPH-cytochrome c reductase activity was about equal in the two stages, andi unaffected by all three pretreatments.

The adult preparations also had a higher G-fold) basal BP monooxygenase activity (Table II) whereas the relative increases after the pretreatmenta were more marked in larvae. PB increased the activity more than S-fold in larvae compared tal 2.6.fold in adults, while the activity was enhanced 49fold and 1.3-fold, respectively, by PCB. The most marked difference was, however, observed in the response to BNF pretreatment, where the activity in larvae was enhanced 2.2.18’old (P < 0.001) while it was totally unaffected by BNF in adult flies.

The demethylation of p-nitroanisole was also considerably (49fold) lower in larval microsomes, while 7-ethoxycoumarin-deethylation was performed with about equal efficiency with the two preparations (Table II). A 2-3.fold increase was caused by PB and 1.3-Sfold by PCB in both adult and larval preparations while BNF caused no or insignificant increases in these two activities.

45

TABLE I

_~_l_l._ll”l_~--~_L”.^.XI--rrril .____~_-.--~ _,__--Lc- .I-LII,---^__- ~_--.---.- --. _ .___ -_ Cytochrome P-450 NADPH-cytochrome

f reductase activity Pre- (nmot/m~ micro- Amnr (nmol cyt. c reducedlmg trentment~ somal protein) (nml microsomal proteinimin

LIWWW? None PB Pi% BNF

Adult flies None 0.19 ‘” 0.01 PB 0.37 t 0.03** PCB 0.33 +- 0.01” BNF 0.22 ? 0.02

0.05 :* 0.0 1

0.16 + 0.01***

0.17 t 0.03*** 0.08 l 0.01

450 22: 1 452 21 r 3 446 29-5 450 26 f 3

450 25-2 450 2523 450 23 * 2 450 2624

“As dcwx%wd in Materials nnd Methods.

The detection of the benzoylated biphenyl metabolites was a very sensitive parameter and the detection limit under the present conditions was abaut

!Mpmol metabolite formed fmg microsomal protein per min. Lower amounts, which could be discerned on the chromatograph but were too low to be calculated with certainty, are classified as trace amounts in Table III. Thtx high ~nsitivity of the analysis made it possible to detect the very low metabolism of biphenyl especially in larvae.

The metabolism of biphenyl gave rise to 3-OH- and 4-OH-biphenyl as the minor and majar metabolites, respectively. The+ o-hydroxylated product, P- ~~-biphenyl was only formed in trace amount by adult flies and it was not at all detectable in larvae. All three pretreatments strongly induced the for- mation of 30 and 4nQH-biphenyl in larval preparations. The 3-OH-biphenyl was in~~ased &fold whereas 4.OH=biphenyl was increased 6-‘7.fold. The total amount of mota~lites formed was about equal after all the pretreat- ments. In adults, where the basal activities were much higher than in larvae. the only significant increase was a doubling of the 4.hydroxylation following PB t~~tment; PC% even caused R marked decrease in the hydsoxylation at ~=~sition.

Figure 3 shows a densitometric tracing after heme staining of microsomes from PUJ.treat& adult flies run on SDS gel electrophoresis. The heme pattern did not differ from control microsomes, but was easier to analyse because of the higher cytochrome P-450 content in t.he PCB-induced pre- paration, Three home containing areas could be seen in the molecular weight region 4 ?, 000-66 000.

TA

BL

E

I!

EF

FE

CT

S

OF

EN

ZY

ME

-IN

DU

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G

AG

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G-M

ET

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G

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S

IN M

ICR

OS

GM

RS

IS

OLA

TED

FR

OM

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R

lEL

AN

ClG

AST

ER

M

AR

SNiiS

6%

) A

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AR

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E

Th

e va

lues

rep

rese

nt

mea

n +

S.E

. of

at

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t 3

expe

rim

ents

. l P <

0.0

5;

**P

< 0

.01:

***

P i

0.

001.

BP

mon

ooxy

gen

ase

7-E

thox

ycou

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p-N

itro

san

isol

e ac

tivi

ty

deet

hyl

ase

acti

vity

de

met

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ase

acti

vity

P

re-

(pm

ollm

g m

icro

som

al

(pm

ol/m

g m

icro

som

al

(nm

ollm

g m

icro

aom

al

trea

tmen

e pr

ob?.

in/m

in)

prot

ein

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) pr

otei

n/m

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Non

e 52

28

4425

0.

11a0

.02

PB

27

4%32

”’ 11

0-t1

3***

0.

27 2

0.0

4**

215k

30””

75

Z6”

0.

34 ?

I 0.0

5* *

B

NF

11

5r4*

**

5726

0.

16 r

0.0

3

Adu

lt t

lies

N

one

2412

17

6757

0.

48 k

0.0

3 P

B

6212

53”’

140

% 1

6**

1.07

2 0.

11**

* P

CB

31

8 2

14*

1445

5””

0.

64 2

0.0

8 B

NF

24

1230

65

212

0.45

2 0

.05

aAs

desc

ribed

in M

ater

ials

an

d M

eth

ods.

47

TABLE III

The ~aherr rcpronent mean ’ $.E. of at least 3 uxpcriments. *P 0.05: * * l P. 0.001; +Significant decreane. --my__

Larvae

~~--” 1_1__- -- _ prc. 2aOH-biphenyl SOW-biphenyl COH-biphenyi treatment. nmol pdUct formed/ml: microsomal protein per min

IIxI~~_~w-P ___.

None trace ~m~unt~b 5zl FB 9 ‘L 2 34 rt 7’ Rx 9r 1 31 f 7* BNF lo? 1 32~8’

Adult flies NOlit? trace amounts 29+I 1192 12 PB trace amounts 30 t 2 263+2S”‘ PCB trnce amount8 14 1) ‘21 118 +_ 17 BNF trace amounts 31-3 156% 16

- ._.

aAs &scribed in Materials and Methods. “Discernible on the CC chromatogram. but betow the detection level ( 2pmollmg/min\ under the exprimental condition8 described.

I Intensity

of slalnlng

Molecular

welghl

Reglen III Rsgton II Ra@lan I

Fig. 3. Heme rontont. of mitroaomeo from PCB&reated adult Karnris 60~ flies. Densitometric tracing wapl ~~f~~~ at 6tMJ nm. The geet WAY stained with 3,3’,~~~-tetramethyl~nzidlne,

In region 1 (42 OQO-Q8OOQ) one could distinguish one peak, in region II (48 OQO-60 000) two peaks Rnd two shoulders and in region III (60 000-66 000)

one braad peek. Tbo home protein8 in region III were located in an area that is usually not associated with the various cytochrome P-450 species in mammals. Heme staining of microaomal preparations from larvae was extremely difficult to perform probably due to the low st.ability and the low P-450 content. The only verified info~ation from repeated experiments was a alight increase in the staining intensity of the 46 00059000 molecular weight region.

Mall!cutnr PI3 weigh1 I,tWViW Adultn

Pretrontmc?W PCB LWVHI~ Adults

BNP Larvae Adults

Protein staining of the gels revealed several bands within the heme- containing area (Fig. 4). Increased protein content in specific bands was observed in microsomes from larvae and adult flies pretreated with PB, PCB or BNF when compared to microsomes from untreated animals. The protein bands referred to in Fig. 4 and Table IV were all located in the molecular weight region 48 000-60 000 and were all increased by at least one inducer in larvae and/or adult flies. Both in larvae and adults PCB and BNF markedly increased the protein content of two bands with apparent molecular weights of 51 600 and 52000; the latter probably consisted of two bands. PCB and BNF also caused minor increases in the bands with molecular weights of 54000 and 56000 in larvae. In adult flies, PB increased the 54 000-. 56000- and 58OOO=protein bands. In larvae, PB pretreatment did not affect these bands, while on the other hand the protein band with an apparent molecular weight of 51500 was increased. It can be noted that the band with a molecular weight of 58000 was markedly weaker in larvae when compared to adult flies. On the gel with microsomes from adult flies (Fig. 4) an increase in a band with an apparent molecular weight of 46000 was also discerned after PCB and BNF treatment. This effect has ucjt been verified in other experiments and must be judged with caution. The increases mentioned in Table IV. even the minor ones, have been reproduced in several experiments.

The rewu1t.s huve demanstrated a much higher P-450 content and a higher capacity for most P-460 mediated reactions in adult flies when compared to larvae. A comparison of various mammalian species also indicated much lower activities of the xenobiotic-metabolizing enzymes in foetal than in adult liver [19,20]. It was therefore interesting to note that the capacity to O-deethylate ‘I-ethoxycoumarin was about equal in both stages of Drosophila

50

studied. This difference in the temporal control of the metabolism indicated the presence of more than one cytochrome P-450 isozymc in Dmsophila. &Vera1 qualitative differences were seen in response to the different pretreatments. PCB was a poor inducer of all the activities studied in adult flies, while in larvae in most cases it was comparable with PB. Especially in the 3-hydroxylat,ion of biphenyl the difference was dramatic, a potent in- duction in larvae compared to a marked decrease in adults after PCB pretreatment. BNF pretreatment was totally inefficient as an inducer of BP monooxygenase activity in adults, while the effect in larvae was statistically significant. Except for an insignificant tendency to increase the formation of 4-OH-biphenyl, no efect at all of BNF pretreatment was observed in adults. in larva? on the oth.er hand 3- and 4-OH-biphenyl as well as BP hydroxy- lation were increased severalfold. A different response to inducers during pre- and postnatal stages has been observed also in mammals [l-31.

Five protein bands were tentatively identified as possible cytochrome P-450 forms (Table IV). The identification was made on the basis of heme content and on inducibility of the specific protein bands. All three pretreat- ments gave rise to such induction, most marked after pretreatment with PCB. The two major BNF-induced forms had considerably lower apparent molecular weights (51500 and 52 000) than have previously been reported for mammals, where pslycyclic aromatic hydrocarbons (PAHl induces P-450 forms with apparent molecular weights of 54000-60 000 [1,21-231. It is worth noting that the marked increase in the protein content of these two BNF-inducible bands was not in accordance with the weak effect of BNF treatment on the enzyme activities studied. The high amount of protein might represent induction of forms not contributing to the cytochrome P-450-depend.ent metabolism studied so far. Inducible proteins other than cytochrome P-450 could also be present, even if the large heme concentration in this area makes it less plausible. Finally, BNF gave rise to minor increases in the two bands with apparent molecular weights of 54 000 and 56000 in larvae but not in adult flies. These changes might reflect the differences in enzyme activities following pretreatment with BNF that were observed between the two stages. PB induced different forms in adult flies and larvae. The larval form had a molecular weight (516001 in the range of the major mammalian PB-induced P-450 [22,23], while the adult forms had higher molecular weights. Little is known about which protein bands might increase following F’B administration in prenatal mammals. The differences between larvae and adult flies in the molecular weights of the PB-inducible bands have so far not been possible to correlate to differences in the metabol- ism between the stages. All the same they supported the indications of deveiopmental modifications of the cytochrome P-450 system. The markedly higher content of the protein with an apparent molecular weight of 58 BOO in adult flies when compared to larvae (Fig. 4) also gave similar evidence.

Naquira et. al. I241 partly purified three forms of cytochrome P-450 from uninduced Drosophila. The apparent molecular weights of 50 800, 51760 and 54 800 reported were in good agreement with the inducible forms tentatively identified here as cytochrome P-450 species (Table IV).

BNF was IC’IIS efficient than expected in enhancing the enzyme activities that ~IW pr~ominantly induced by PAH in mammals, namely afyl hydra. carbon hy&oxylu~ and bipbenyl %hydroxylase [22,23,25,26]. No blue shift

option maximum of the USbound difference spectra was observed (T&e XI and the BNF-induced enzymes in larvae were insensitive to in- hibition by ~rgna~hth~f~avon~ IS]. Together, these data supports the assump- tion that the induction of enzymes, corresponding to the two different cytochromct B-450 species induccvi by BNF in mammals [21], is low or absent in ~~~i~~. The induction of the enzymes mentioned above in mammals is de~ndent on a cytosolic receptor for PAH, the tetraehloro-dibenz~p~oxin (TCDD) receptor [Nl and genetically regulated defects in the receptor pro- tein lead& to non-rc%ponsiveness to BNF and 3=methylcholanthrene induction i27,28]. So far. the at~mpts to identify the TCDD receptor in ~roso~~~~a larvae have not been succeseful (H~llstriim arid Carlstedt-Duke, unpublished results). This possibly supports the presence of a different or defective receptor in Rrmmphila. However failure in detection due to proteolysis of an unstable protein receptor in the whole-buy preparation can not be ruled out.

The formation of 2-OH=biphenyl was extremely iow both in larvae and in adult flies. Jordan and Smith [29] detected 4.OH-biphenyl but not 2-OH- biphenyl formation also by house fly homogenates. In rabbits [l] the for- mation of the 2-OH~rneta~~‘ce could not be demonstrate in adults. In contrast to our findings, the earlier developmental stages had a detectable formation. The basal and PAH-induced 2-hydroxylation of biphenyl was shown to be closely correlated to cytochrome PI-450 in the mouse [30], thr enzyme also responsible for the formation of the 7,8-dihydrodiol from BP 1211. A similar enzymatic correlation was indictiiced in lzrosophilu, as the luw basal activity and response to BNF-pretreatment of the biphenyl Z-hydroxy- lase reported here corresponded to a very iow formation of BP-7.8.diol by microsomes from BNF-treated larvae [81. A low content of the Y,-450.type enzyme leading to a low formation of mutagenic bay-region dihydrodiol epoxides 121,311 might explain the weak mutagenic effect of PAW in Droso- China f&32] as previously discussed [$I.

The cytochrome B-450 content and BP monoosygenuse activity in larvae from th@ isogenis strain of Karsniis 60, used in this study were in good agreement with the data for the parent wild type strain previously reported [S]. The indue~bility of the BP mo~~xygenase act‘<ity by PB and BNF seemed, however, lower in the isogenic sirai-. suggesting that genetic differences may have been created during the isohrenization process due to a ~~n~ti~ drift. Such a genetic drift in the small laboratory animal populations might be an im~rtant factor in int~rl~boraLo~ variation in metabolism and mutnRonis activity in Dmophilu and other species.

The higher metabolic capacity of most cytochromc P-45O-dependent enzyme ~~ctivities in adult flies indicates that treatment of adult fiies should result, in a higher sensitivity to det.ect indirect mutagens and carcinogens than larval treatment. The formation of 2-OH-biphenyl was however equally weak in both stages, indicating that the sensitivity to PAI-I would be bad in both stales, Factors sue+ as uptake and distribution of the test compound

might also influence the relative detection capacity in tests with adult flies or larvae. Pretreatment with inducers increases several enzyme activities lead- ing to higher sensitivity to some classes of indirect mutagens. BNF pretreatnkent, however, failed to enhance the formation of 2=OH=biphenyl. This indicated that the &-45%type enzyme in ~~so~~~~ is non~s~nsive La BNF and other PARS, and pretreatment with this class of inducer should not be expected to increase the formation of mutagenic metabolites from PAH. Thus PAH induction is not a way to increase the detection capacity for carcinogenic PAHs, which are not or are only weakly mutagenic in Dr~as- phila tests.

In the Ames’ test on Salmonella typhimurium, the rats frlrtm which the liver S9 fraction, used as metabolizing system, is prepared are routinely pretreat& with PCB. This pretreatment can not be used in Drosophir’a recessive 1ethLtl tests because of the Iweak inducing potency of PCB in adult flies (Tables I-IV) when compared to the potency in rat liver. An increased mutagenic effect after ~B-t~atment of the flies could occur in some cases, but the pretreat= ment might sometimes be inefficient or even decrease the mutagenic effect. Thus it cannot be recommended for the testing of samples, for which the route of activation is unknown. Taken together, enzyme induction is not a general way of increasing the detection capacity and sensitivity of mutagenicity tests with Drosophihz as the test organism.

ACKNOWLEDGEMENTS

The authors wish to thank Dr. R. Toftg&rd and Professor C. Ramel for helpful comments and c$ticism during manuscript preparation. Thanks are also due to Professor J.-A. Gustafsson and Professor S. Jensen for discussion and for the use of their laboratory facilities and to Dr. L. Rennberg for technical aids. We are also much obliged to Miss M. Lienzen for excellent technical assistance and encouragement. The work was supported by the Swedish Council for Planning and Coordination of Research.

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