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Noninducibility of cytochrome P-450 in the earthworm Dendrobaena veneta
Cony. Eiochem. Physid. Printed in Great Britain
Vol. 8X,
No. I, pp. 85-87,
1986
0306-4492/86
$3.00 + 0.00 Ltd
Pergamon Journals
NONINDUCIBILITY OF CYTOCHROME P-450 IN THE EARTHWORM DENDROBAENA K?SV,!VA DANIEL L. MILLIGAN,*JOHN G. BABIsHt and EDWARDF. NEUHAUSER*$ *Department of Agricultural Engineering, Cornell University, Ithaca, NY 14853, USA and TDepartment of Preventive Medicine, New York State College of Veterinary Medicine. Cornell University, Ithaca, NY 14853, USA (Receiwd 3 January 1986) Abstract-l. Cytochrome P-450 has been measured in the earthworm Dendrobaena ueneta (Rosa) in a direct spectrophotometric procedure. 2. The P-450 was found not in the dense microsomal fraction. but in the less dense overlying fraction often referred to as buffy coat. 3. Earthworm P-450 was not induced by 3-methylcholanthrene or phenobarbital.
INTRODtJCTlON Studies have shown that many invertebrates and vertebrates are able to oxidatively metabolize chemicals that are foreign to their normal metabolism (Smith, 1977; Machinist et al., 1968). These chemicals, often called xenobiotics, may be natural or man-made. Some examples of xenobiotics are secondary plant metabolites, drugs and pesticides. The ability of an organism to survive in the presence of toxic xenobiotics is directly related to its ability to biotransform and excrete these substances. One measure of this ability is the presence and inducibility of enzymes that metabolize xenobiotics. The nature of these enzymes in the earthworm is the subject of this study. Many studies have been performed concerning the toxicity of chemicals to earthworms (Edwards and Thompson, 1973; Thompson, 1973), but relatively few have focused on the metabolism of these chemicals or on the enzymes involved in this metabolism (Nelson et al., 1976; Stenersen et al., 1979). In many species of vertebrates and invertebrates, a group of monooxygenases termed cytochrome P-450 (Omura and Sato, 1964) have been implicated in the oxidation of xenobiotics (Kato, 1979). A recent review by Stenersen (1984) reveals that most of the studies of detoxification processes in earthworms have focused on the isolation of oxidation products in ho. Attempts to identify the enzymes involved in this process have been complicated by the presence of interfering pigments. However, the presence of cytochrome P-450 in Lumbricus terrestis has recently been demonstrated by Liimatainen and Hanninen (1982) after separation of the protein by column chromatography. In this laboratory direct measurements of cytochrome P-450 have been made in microsomes prepared by homogenization and centrifugation of the gut wall of Dendrobaena veneta (Rosa). This procedure has been used to study the effects of two classical inducers of xenobiotic metabolizing systems, 3-methylcholanthrene (3-MC) and phenobarbitol fAuthor
to whom
correspondence
should
be sent. 85
(PB), on the levels of P-450 in earthworms. Knowledge of the inducibility of these systems will yield a better understanding of the ability of earthworms to adapt to perturbations in the environment. MATERIALSAND
METHODS
Earthworms used in the study were adult D. ueneta, approx. 6 months old. They were raised on a constant organic food source at 20°C to a weight of 335 g. In an attempt to ascertain the ability of the earthworm to adapt to a changing environment, adult earthworms were exposed to chemicals that are known to induce cytochrome P-450 in other species. 3-MC (75 mg/kg in 50 ~1 corn oil) or PB (75 mg/kg in 50 ~1 dH,O) was injected into groups of l&15 worms, while controls were injected with corn oil or dH,O alone. 3-MC was given in one dose and microsomes were prepared 72 hr after injection. PB was given every other day for 4 days and microsomes were prepared 24 hr after the last dose. The preparation of microsomes follows the method developed for armyworms by Crankshaw et al. (1979). Worms were allowed to void for 48 hr in a shallow dish of distilled water. Any remaining contents of the gut were then forced out by rinsing with ice cold 0.25 M sucrose containing 1mM EDTA, 1% polyvinylpyrrolidone and 2 mM phenylmethane sulfonyl fluoride, pH 7.5. The gut tissue was removed and gently homogenized (I:4 w/v) with a PotterElvehjen homogenizer and a Teflon pestel in the same medium. The homogenate was centrifuged at YOOOg for 15min in a Beckman Model 52-21 centrifuge and the supernatant fraction was decanted through glass wool. The supernatant fraction was centrifuged in a Beckman Preparative Ultracentrifuge with a type 30 rotor at 100,OOOg for 60min. The supernatant fraction was removed and the remaining dense red pellet and the overlying fluffy red layer were resuspended in 3 vol. of fresh medium and recentrifuged as before for 30 min. Again, the supernatant fraction was removed and all of the pelleted material along with the buffy coat was resuspended to a 1: I (w/v) dilution of original gut wet wt to 50 mM Tris-Hdl, 30% glycerol, pH 7.5. All procedures were uerformed at tF4”C. One milliliter aliquots were frozen in liquid nitrogen and stored at -70°C. Cytochrome P-450 was determined by the method of Orrenius e( al. (1973) using the Aminco DW-2C scanning spectrophotometer. Microsomes were thawed on ice and diluted to 1 mg protein/ml with Tris-glycerol buffer. NADH (0.1 PM) was added to a 2 ml aliquot of the diluted
86
DANIEL L. MILLIGAN et al. Table
0.05,
1..
‘.
.
.
‘1..
.
‘.
nm
microsomes and CO was bubbled through the solution for 30 sec. This served to block the interference of cytochrome b, and hemoglobin in the microsomes. This solution was
split between sample and reference cuvettes and several crystals of Na,O,S, were added to reduce cytochrome P-450. A molar extinction coefficient of 91 mM-’ cm-’ was used for quantitating P-450 levels (Omura and Sato, 1964). Protein content was determined by the method of Sutherland et al. (1949).
RESULTS AND DISCUSSION
Cytochrome
P-450 was identified in the gut of D. spectophotometric methods. Earthworm P-450 was not found in the same fraction as in microsomal preparations of vertebrate liver. This may explain the past failures of procedures involving centrifugation to indicate the presence of P-450 in earthworms (Nelson et al., 1976; Liimatainen et al., 1982). In liver preparations, P-450 is found in the densely packed microsomal fraction after the 100,000 g spin. In our earthworm preparations P-450 cannot be measured in this fraction, but can be observed in the fluffy overlying fraction often referred to as buffy coat. As seen in Fig. 1, the spectrum of cytochrome P-450 includes a peak at 424nm. Since the contribution of hemoglobin to the spectrum is cancelled by the addition of CO to both sample and reference veneta by direct
Table 2. The effect of inducers
PB’ CO”trOl
0.405 0.582
2.352 2.106
3-MC+ Control
0. I56 0. I59
0.747 0.655
0.220 0.164
0.918 0.630
soil:
cuvettes, the peak at 424nm is probably due to cytochrome b,. Other researchers have reported low levels of reductase activity in microsomal preparations from marine organisms such as the lobster (James et al., 1982). If this is the case with the earthworm, the added NADH may not reduce the b, adequately in both the sample and reference cuvettes. With the addition of dithionite to the sample cuvette, additional b, would become reduced leading to the appearance of a peak at 424 nm. The addition of only dithionite in the sample cuvette produced the same peak at 424 nm, giving further evidence that this peak was due to a &-type cytochrome. No apparent differences in P-450 levels were found between control earthworms and those exposed to 75 mg/kg 3-MC or PB (Table 1). One possible explanation for the apparent noninducibility of P-450 is that the earthworms are already maximally induced by the organic food source on which they are raised. However, in a preliminary study, P-450 levels did not change when worms were kept for 3 weeks on a mixture of sand, peat moss and kaolin and were fed powdered rabbit food. In responsive animals the effect of inducers on levels of P-450 is pronounced. Table 2 summarizes the inducibility of P-450 in a variety of vertebrate and invertebrate species. Responsive organisms typically show two- to three-fold increases in P-450 upon induction, while nonresponsive animals show little or
on the levels of cytochrome
P-450 in a variety of animal
Treatment
Rat
Control PB’ 3-MC+
0.6GO.87 1.28-1.46 1.28-1.37
Dent
Sheepshead
Control 3-MC
0.27-0.41 0.5 1-I .09
James and Bend (1980)
Housefly
Control PB
2.00 2.95
Perry et al. (1971)
Control BooPf Aroclor
0.08 0.14 0.20
Lee (1981)
Armywonn
Control PM95 HMBII
1.22 3.65 2.67
Brattsten
Spiny lobster
Control 3-MC
2.08 1.55
James and Little (1984)
Polychaete
worm
*PB, phenobarbital. +3-MC, 3methylcholanthrene. $BcoP, benzo[a)pyrene. @PMB, pentamethylbenzene. //HMB, hexamethyl benzene.
aeneta.15
“mole P-450 per g gut tissue
Cytochrome P-450 (nmol/mg protein)
Species
D.
‘Phenobarbital, 75 mg/kg in 50 11 dH,O. + 3-Methylcholanthrene, 75 mg/kg in 50 /I I corn oil. fEarthworms were kept on a mixture of sand, peat moss and kaolin for 3 weeks. The food source was powdered rabbit food.
of D. ueneta gut
1. Dithionitedifference spectrum microsomes.
P-450 in the earthworm, worms/determination “mole P-450/mg protein
Artificial Control
1
495
450
400
Fig.
.
I. Cytocbrome
species
Reference
et al. (1980)
and Wilkenson
(1973)
Cytochrome P-450 in the earthworm no change in enzyme levels. If noninducibility of cytochrome P-450 is a general characteristic of earthworms, then one would expect them to accumulate nonpolar xenobiotics under conditions where constitutive levels of the enzymes are unable to metabolize the chemicals present in the body, This would increase the earthworm’s susceptibility to toxic xenobiotics and increase the probability that the accumulated xenobiotic would move up the food chain.
87
Lee R. F. (1981) Mixed function oxidases (MFO) in marine invertebrates. Mar. Bioi. Lett. 2, 87-105. Liimatainen A. and Hanninen 0. (1982) Occurrence of cytochrome P-450 in the earthworm Lumbrieus terrestris. In Cy~ochrome P-450: Biochemistry, Rio~hysies and Environmental Im~fi~at~ons (Edited by Hietanen E., Lartinen M. and Manninen M.), pp. 255-258. Elsevier, Amsterdam. Machinist J. M., Dehner E. W. and Ziegler D. M. (1968) Microsomal oxidases, III. Comparison of species and organ distribution of dialkylarylamine N-oxide dealkylase and dialkyiarylamine N-oxidase. Arch. Ifioehem. Biophys. 125, 858-864.
REFERENCES
Brattsten L. B. and Wilkinson C. F. (1973) Induction of microsomal enzymes in the southern armyworm (Prodenia eridania). Pestic. Biochern. Physiol. 3, 3933407.
Crankshaw D. L., Hetnarski H. K. and Wilkinson C. F. (1979) Microsomal NADPH~yto~hrome c reductase from the midgut of the southern armyworm (Prodenia eridania). Insect Biochem. 9, 43-48. Dent J. G.. Graichen M. E.. Schnell S. and Lasker J. (1980) Constitutive and inducedhepatic microsomal cytochromk P-450 monooxygenase activities in male Fischer-344 and CD rats. A comparative study. Toxic. appl. Pharmac. 52, 45-53.
Edwards C. A. and Thompson A. R. (1973) Pesticides and the soil fauna. Residue Rev. 45, l-80. James M. 0. and Bend J. R. (1980) Polycyclic aromatic hydrocarbon induction of cytochrome P-450 dependent mixed function oxidases in marine fish. Toxic. appl. Pharmac. 54, 117-133.
James M. 0. and Little P. J. (1984) 3-Methylcholanthrene does not induce in viuo xenobiotic metabolism in spiny lobster hepatopancreas, or affect in o&o deposition of benzo[a]pyrene. Camp. bioehem. Physiof. 78C, 241-245. James M. O., Sherman B., Fisher S. A. and Bend J. R. (1982) Benzo[a]pyrene metabolism in reconstituted monooxygenase systems containing cytochrome P-450 from lobster (Homarus americanus) hepatopancreas fractions and NADPH cytochrome P-450 reductase from pig liver. Bull. Mt Desert Isl. biol. Lab. 22, 37-39. Kato R. (1979) Characteristics and differences in the hepatic mixed-function oxidases of different species. Pharmac. Ther. 6, 41-98.
Nelson P. A.. Stewart R. R., More% M. A. and Nakatsugawa T. (1976) Aldrin epoxidation in earthworm Lumbricus terrestris L. Pestic. Biochem. Physiol. 6, 243-253.
Omura T. and Sato R. (1964) The carbon monoxide-binding pigment of liver microsomes, II. SoIubilization, purification and properties. f. biol. Chem. 239, 2379-2385. Orrenius S., Ellin A., Jakobsson S. V., Thor H., Cinti D. L., Schenkman J. B. and Estabrook R. W. (1973) The cytochrome P-450 containing monooxygenase system of rat kidney cortex microsomes. Drug Metab. Disp. 1, 35G357.
Perry A. S., Dale W. E. and Buckner A. J. (1971) Induction and repression of microsomal mixed function oxidases and cytochrome P-450 in resistant and susceptible houseflies. Pestic. Biochem. Physioi. 1, 131-142. Smith J. N. (i977) Comparative detoxication of invertebrates. In Drug Metabolism: From Microbe to Man (Edited bv Parke D. V. and Smith R. L.). I, DD. I. 219-232. Taylor and Francis, London. Stenersen J., Guthenberg C. and Mannervik B. (1979) Glutathione $-transferase in earthworms (Lumbricidae). ~jochem. .I. 181,47-50.
Stenersen J. (1984) Detoxication of xenobiotics by earthworms. Camp. Biochem. Physioi. 78C, 249-252. Sutherland E. W., Cori C. F., Haynes R. and Olsen N. S. (1949) Purification of the hyperglycemic-glycogenolytic factor from insulin and from gastric mucosa. J. biol. Chem. 180, 8255837. Thompson A. R. (1973) Pesticide residues and soil invertebrates. In Environmental Pollution by Pesticides (Edited by Edwards C. A.), pp. 87-113. Plenum Press, New York.
Vol. 8X,
No. I, pp. 85-87,
1986
0306-4492/86
$3.00 + 0.00 Ltd
Pergamon Journals
NONINDUCIBILITY OF CYTOCHROME P-450 IN THE EARTHWORM DENDROBAENA K?SV,!VA DANIEL L. MILLIGAN,*JOHN G. BABIsHt and EDWARDF. NEUHAUSER*$ *Department of Agricultural Engineering, Cornell University, Ithaca, NY 14853, USA and TDepartment of Preventive Medicine, New York State College of Veterinary Medicine. Cornell University, Ithaca, NY 14853, USA (Receiwd 3 January 1986) Abstract-l. Cytochrome P-450 has been measured in the earthworm Dendrobaena ueneta (Rosa) in a direct spectrophotometric procedure. 2. The P-450 was found not in the dense microsomal fraction. but in the less dense overlying fraction often referred to as buffy coat. 3. Earthworm P-450 was not induced by 3-methylcholanthrene or phenobarbital.
INTRODtJCTlON Studies have shown that many invertebrates and vertebrates are able to oxidatively metabolize chemicals that are foreign to their normal metabolism (Smith, 1977; Machinist et al., 1968). These chemicals, often called xenobiotics, may be natural or man-made. Some examples of xenobiotics are secondary plant metabolites, drugs and pesticides. The ability of an organism to survive in the presence of toxic xenobiotics is directly related to its ability to biotransform and excrete these substances. One measure of this ability is the presence and inducibility of enzymes that metabolize xenobiotics. The nature of these enzymes in the earthworm is the subject of this study. Many studies have been performed concerning the toxicity of chemicals to earthworms (Edwards and Thompson, 1973; Thompson, 1973), but relatively few have focused on the metabolism of these chemicals or on the enzymes involved in this metabolism (Nelson et al., 1976; Stenersen et al., 1979). In many species of vertebrates and invertebrates, a group of monooxygenases termed cytochrome P-450 (Omura and Sato, 1964) have been implicated in the oxidation of xenobiotics (Kato, 1979). A recent review by Stenersen (1984) reveals that most of the studies of detoxification processes in earthworms have focused on the isolation of oxidation products in ho. Attempts to identify the enzymes involved in this process have been complicated by the presence of interfering pigments. However, the presence of cytochrome P-450 in Lumbricus terrestis has recently been demonstrated by Liimatainen and Hanninen (1982) after separation of the protein by column chromatography. In this laboratory direct measurements of cytochrome P-450 have been made in microsomes prepared by homogenization and centrifugation of the gut wall of Dendrobaena veneta (Rosa). This procedure has been used to study the effects of two classical inducers of xenobiotic metabolizing systems, 3-methylcholanthrene (3-MC) and phenobarbitol fAuthor
to whom
correspondence
should
be sent. 85
(PB), on the levels of P-450 in earthworms. Knowledge of the inducibility of these systems will yield a better understanding of the ability of earthworms to adapt to perturbations in the environment. MATERIALSAND
METHODS
Earthworms used in the study were adult D. ueneta, approx. 6 months old. They were raised on a constant organic food source at 20°C to a weight of 335 g. In an attempt to ascertain the ability of the earthworm to adapt to a changing environment, adult earthworms were exposed to chemicals that are known to induce cytochrome P-450 in other species. 3-MC (75 mg/kg in 50 ~1 corn oil) or PB (75 mg/kg in 50 ~1 dH,O) was injected into groups of l&15 worms, while controls were injected with corn oil or dH,O alone. 3-MC was given in one dose and microsomes were prepared 72 hr after injection. PB was given every other day for 4 days and microsomes were prepared 24 hr after the last dose. The preparation of microsomes follows the method developed for armyworms by Crankshaw et al. (1979). Worms were allowed to void for 48 hr in a shallow dish of distilled water. Any remaining contents of the gut were then forced out by rinsing with ice cold 0.25 M sucrose containing 1mM EDTA, 1% polyvinylpyrrolidone and 2 mM phenylmethane sulfonyl fluoride, pH 7.5. The gut tissue was removed and gently homogenized (I:4 w/v) with a PotterElvehjen homogenizer and a Teflon pestel in the same medium. The homogenate was centrifuged at YOOOg for 15min in a Beckman Model 52-21 centrifuge and the supernatant fraction was decanted through glass wool. The supernatant fraction was centrifuged in a Beckman Preparative Ultracentrifuge with a type 30 rotor at 100,OOOg for 60min. The supernatant fraction was removed and the remaining dense red pellet and the overlying fluffy red layer were resuspended in 3 vol. of fresh medium and recentrifuged as before for 30 min. Again, the supernatant fraction was removed and all of the pelleted material along with the buffy coat was resuspended to a 1: I (w/v) dilution of original gut wet wt to 50 mM Tris-Hdl, 30% glycerol, pH 7.5. All procedures were uerformed at tF4”C. One milliliter aliquots were frozen in liquid nitrogen and stored at -70°C. Cytochrome P-450 was determined by the method of Orrenius e( al. (1973) using the Aminco DW-2C scanning spectrophotometer. Microsomes were thawed on ice and diluted to 1 mg protein/ml with Tris-glycerol buffer. NADH (0.1 PM) was added to a 2 ml aliquot of the diluted
86
DANIEL L. MILLIGAN et al. Table
0.05,
1..
‘.
.
.
‘1..
.
‘.
nm
microsomes and CO was bubbled through the solution for 30 sec. This served to block the interference of cytochrome b, and hemoglobin in the microsomes. This solution was
split between sample and reference cuvettes and several crystals of Na,O,S, were added to reduce cytochrome P-450. A molar extinction coefficient of 91 mM-’ cm-’ was used for quantitating P-450 levels (Omura and Sato, 1964). Protein content was determined by the method of Sutherland et al. (1949).
RESULTS AND DISCUSSION
Cytochrome
P-450 was identified in the gut of D. spectophotometric methods. Earthworm P-450 was not found in the same fraction as in microsomal preparations of vertebrate liver. This may explain the past failures of procedures involving centrifugation to indicate the presence of P-450 in earthworms (Nelson et al., 1976; Liimatainen et al., 1982). In liver preparations, P-450 is found in the densely packed microsomal fraction after the 100,000 g spin. In our earthworm preparations P-450 cannot be measured in this fraction, but can be observed in the fluffy overlying fraction often referred to as buffy coat. As seen in Fig. 1, the spectrum of cytochrome P-450 includes a peak at 424nm. Since the contribution of hemoglobin to the spectrum is cancelled by the addition of CO to both sample and reference veneta by direct
Table 2. The effect of inducers
PB’ CO”trOl
0.405 0.582
2.352 2.106
3-MC+ Control
0. I56 0. I59
0.747 0.655
0.220 0.164
0.918 0.630
soil:
cuvettes, the peak at 424nm is probably due to cytochrome b,. Other researchers have reported low levels of reductase activity in microsomal preparations from marine organisms such as the lobster (James et al., 1982). If this is the case with the earthworm, the added NADH may not reduce the b, adequately in both the sample and reference cuvettes. With the addition of dithionite to the sample cuvette, additional b, would become reduced leading to the appearance of a peak at 424 nm. The addition of only dithionite in the sample cuvette produced the same peak at 424 nm, giving further evidence that this peak was due to a &-type cytochrome. No apparent differences in P-450 levels were found between control earthworms and those exposed to 75 mg/kg 3-MC or PB (Table 1). One possible explanation for the apparent noninducibility of P-450 is that the earthworms are already maximally induced by the organic food source on which they are raised. However, in a preliminary study, P-450 levels did not change when worms were kept for 3 weeks on a mixture of sand, peat moss and kaolin and were fed powdered rabbit food. In responsive animals the effect of inducers on levels of P-450 is pronounced. Table 2 summarizes the inducibility of P-450 in a variety of vertebrate and invertebrate species. Responsive organisms typically show two- to three-fold increases in P-450 upon induction, while nonresponsive animals show little or
on the levels of cytochrome
P-450 in a variety of animal
Treatment
Rat
Control PB’ 3-MC+
0.6GO.87 1.28-1.46 1.28-1.37
Dent
Sheepshead
Control 3-MC
0.27-0.41 0.5 1-I .09
James and Bend (1980)
Housefly
Control PB
2.00 2.95
Perry et al. (1971)
Control BooPf Aroclor
0.08 0.14 0.20
Lee (1981)
Armywonn
Control PM95 HMBII
1.22 3.65 2.67
Brattsten
Spiny lobster
Control 3-MC
2.08 1.55
James and Little (1984)
Polychaete
worm
*PB, phenobarbital. +3-MC, 3methylcholanthrene. $BcoP, benzo[a)pyrene. @PMB, pentamethylbenzene. //HMB, hexamethyl benzene.
aeneta.15
“mole P-450 per g gut tissue
Cytochrome P-450 (nmol/mg protein)
Species
D.
‘Phenobarbital, 75 mg/kg in 50 11 dH,O. + 3-Methylcholanthrene, 75 mg/kg in 50 /I I corn oil. fEarthworms were kept on a mixture of sand, peat moss and kaolin for 3 weeks. The food source was powdered rabbit food.
of D. ueneta gut
1. Dithionitedifference spectrum microsomes.
P-450 in the earthworm, worms/determination “mole P-450/mg protein
Artificial Control
1
495
450
400
Fig.
.
I. Cytocbrome
species
Reference
et al. (1980)
and Wilkenson
(1973)
Cytochrome P-450 in the earthworm no change in enzyme levels. If noninducibility of cytochrome P-450 is a general characteristic of earthworms, then one would expect them to accumulate nonpolar xenobiotics under conditions where constitutive levels of the enzymes are unable to metabolize the chemicals present in the body, This would increase the earthworm’s susceptibility to toxic xenobiotics and increase the probability that the accumulated xenobiotic would move up the food chain.
87
Lee R. F. (1981) Mixed function oxidases (MFO) in marine invertebrates. Mar. Bioi. Lett. 2, 87-105. Liimatainen A. and Hanninen 0. (1982) Occurrence of cytochrome P-450 in the earthworm Lumbrieus terrestris. In Cy~ochrome P-450: Biochemistry, Rio~hysies and Environmental Im~fi~at~ons (Edited by Hietanen E., Lartinen M. and Manninen M.), pp. 255-258. Elsevier, Amsterdam. Machinist J. M., Dehner E. W. and Ziegler D. M. (1968) Microsomal oxidases, III. Comparison of species and organ distribution of dialkylarylamine N-oxide dealkylase and dialkyiarylamine N-oxidase. Arch. Ifioehem. Biophys. 125, 858-864.
REFERENCES
Brattsten L. B. and Wilkinson C. F. (1973) Induction of microsomal enzymes in the southern armyworm (Prodenia eridania). Pestic. Biochern. Physiol. 3, 3933407.
Crankshaw D. L., Hetnarski H. K. and Wilkinson C. F. (1979) Microsomal NADPH~yto~hrome c reductase from the midgut of the southern armyworm (Prodenia eridania). Insect Biochem. 9, 43-48. Dent J. G.. Graichen M. E.. Schnell S. and Lasker J. (1980) Constitutive and inducedhepatic microsomal cytochromk P-450 monooxygenase activities in male Fischer-344 and CD rats. A comparative study. Toxic. appl. Pharmac. 52, 45-53.
Edwards C. A. and Thompson A. R. (1973) Pesticides and the soil fauna. Residue Rev. 45, l-80. James M. 0. and Bend J. R. (1980) Polycyclic aromatic hydrocarbon induction of cytochrome P-450 dependent mixed function oxidases in marine fish. Toxic. appl. Pharmac. 54, 117-133.
James M. 0. and Little P. J. (1984) 3-Methylcholanthrene does not induce in viuo xenobiotic metabolism in spiny lobster hepatopancreas, or affect in o&o deposition of benzo[a]pyrene. Camp. bioehem. Physiof. 78C, 241-245. James M. O., Sherman B., Fisher S. A. and Bend J. R. (1982) Benzo[a]pyrene metabolism in reconstituted monooxygenase systems containing cytochrome P-450 from lobster (Homarus americanus) hepatopancreas fractions and NADPH cytochrome P-450 reductase from pig liver. Bull. Mt Desert Isl. biol. Lab. 22, 37-39. Kato R. (1979) Characteristics and differences in the hepatic mixed-function oxidases of different species. Pharmac. Ther. 6, 41-98.
Nelson P. A.. Stewart R. R., More% M. A. and Nakatsugawa T. (1976) Aldrin epoxidation in earthworm Lumbricus terrestris L. Pestic. Biochem. Physiol. 6, 243-253.
Omura T. and Sato R. (1964) The carbon monoxide-binding pigment of liver microsomes, II. SoIubilization, purification and properties. f. biol. Chem. 239, 2379-2385. Orrenius S., Ellin A., Jakobsson S. V., Thor H., Cinti D. L., Schenkman J. B. and Estabrook R. W. (1973) The cytochrome P-450 containing monooxygenase system of rat kidney cortex microsomes. Drug Metab. Disp. 1, 35G357.
Perry A. S., Dale W. E. and Buckner A. J. (1971) Induction and repression of microsomal mixed function oxidases and cytochrome P-450 in resistant and susceptible houseflies. Pestic. Biochem. Physioi. 1, 131-142. Smith J. N. (i977) Comparative detoxication of invertebrates. In Drug Metabolism: From Microbe to Man (Edited bv Parke D. V. and Smith R. L.). I, DD. I. 219-232. Taylor and Francis, London. Stenersen J., Guthenberg C. and Mannervik B. (1979) Glutathione $-transferase in earthworms (Lumbricidae). ~jochem. .I. 181,47-50.
Stenersen J. (1984) Detoxication of xenobiotics by earthworms. Camp. Biochem. Physioi. 78C, 249-252. Sutherland E. W., Cori C. F., Haynes R. and Olsen N. S. (1949) Purification of the hyperglycemic-glycogenolytic factor from insulin and from gastric mucosa. J. biol. Chem. 180, 8255837. Thompson A. R. (1973) Pesticide residues and soil invertebrates. In Environmental Pollution by Pesticides (Edited by Edwards C. A.), pp. 87-113. Plenum Press, New York.