Intemt&m, 10 (f97S) 29H99 ICI ScientilicPubliiing Company, A
- Pfinfd in
ALTERATKINS JN HEPATIC DRUG METABOLISM AND LfPID PEROXIDATION DURING ADMlNlSTRATION OF BAYGON, A P CIDE
SURIN~~R J. MAKWUA AND SITARAM S. PAWAR*
(ReceivedJuly 9th. 1974) (Revision receivedSeptember18th, 1974)
8UMMARY
Biochemical studies during low- and high-dose administration of Raygon (a pesticide) to young male rats were performed. It was observed that the activities of drug=me~boIi~ng enzymes were decreased even at a low dose of Baygon and the decrease was much more significant during high-dose injections. Lipid peroxidation was increased with a low dose and the increase was much more pronounced with a high dose. Besides these changes the animals showed physical changes such as salivation, fasciculations, etc. The present of conjugated diene absorption patterns and malona~dehyde formation indicts the in viva Iipid ~roxidation due to Baygon administration. Toxic effects leading to death were noted when the animals were injected with a high dose of the pesticide, Baygon above 25 mg/kg.
INTRODUCTION
Phenyl-substituted methyicarbamates are pesticides of the anticholinesterase type. Several investigators have studied both the in viuo and in vitro metabolism of methyi carbamates in rats l-4, Rao and Anderss and Stevens et at.6 have reported a slight decrease in drug metabolism due to carbaryl in rats and mouse. Baygon being a common pesticide in India, there are several instances in which human beings come across this readily available drug as a means for suicide. The accidental exposure of laymen to this drug is also very common. * To whom all correspondenceshould be addressed. Abbreviation: TPNH, triphosphopyridinenucleotide, reduced form.
Bioch:emical studies during the low and high dose administration of this drug might be useful to establish its role in hepatic drug metabolism. The data regarding this aspect is not available and hence the present communication describes the effect of Baygon on the liver microsomal drug-metabolizing enzyme system and lipid ~roxidation. MATERIALS AND METHODS
C.F. strain young male albino rats initially weighing 55-65 g were obtained from Calcutta. The animals were supplied with rat pellets (Hindustan Lever Ltd., Bombay) and water ad 2%. for 15 days before the experiments began. In the injection experiments, Baygon (a gift from Bayer India Ltd., Bombay) was administered by intraperitoneal injection using corn oil as a carrier. The control animals received a placebo. Injections at a dose level of 5 mg/kg low and 25 mg/kg high were made for two days. During the injection period the animals were supplied food and water ad I& and it was observed that rats from both control and experimental groups consumed the same amounts. The animals were decapitated 24 h after the last injection. The livers were excised, minced with scissors, rinsed with 0.9 % ice-cold saline and homogenized (1: 10 w/v) in 0.25 Nice-cold sucrose with a teflon pestle and glass homogenizer. The homogenates were centrifuged at 9000 x g for 20 min in a refrigerated centrifuge (Remi K-24). The 9000 x g supernatant protein content was estimated according to the Biuret method?. The activities of aminopyrine N-demethylase and acetanilide hydroxylase were assayed according to Schenkman et ~1.8 in a medium containing 50 mM Tris-HCI buffer (PI-I 7.4), 5 m&Z MgCls, 8 mM substrates ~aminopyrine and a~taniiide), 0.2 mg TPNH and 2 mg/ml9000 x g supernatant protein in a final volume of 5 ml. All incubations were carried out in air at 37” in a Dubnoff metabolic shaker and terminated by the addition of 10% ice-cold trichloroacetic acid. The formaldehyde formed using aminopyriue as substrate was estimated according to the procedure of Nashs. The ~-hydroxyace~nilide produced during the oxidation of acetanilide was measured according to the method of Weisburger and Goodallie. TPNH-linked lipid peroxidation was assayed as described by Ernster and Nordenbrandll. Non-enzymatic lipid peroxidation was assayed according to Zalkin and Tappei 13. The malonaldehyde produced was measured according to Betnheim et aLf3.
The liver lipids were extracted according to the method of Folch et al.14, and UV spectra were determined to establish the presence of malonaldehyde at 260-265 nm and diene conjugation band at 230-235 nm. RESULTS AND DiSCUSSKbN
Acute treatment with Baygon elicited a slight but significant increase in liver weights whereas chronic treatment decreased the liver weights slightly. The relative liver weights were slightly increased with low dose, however, a significant increase
297 was observed with high dose. The 9000 x g su~rna~nt protein content was slightly increased with low dose and slightly decreased with higb dose of Baygon. The decrease in aminopyrine N-demethylase activities were 9.7 and 19.7% with low and high dose administration of Baygon, respectively. The decrease in acetanilide hydroxylase activity was only 1.6% with low doses, but 22.3% when the animals were given a high dose. Low doses of Baygon resulted in a significant increase in enzymatic and slight increase in non-enzymatic lipid peroxidation. The increase in lipid peroxidation was more significant with high dose. The liver lipid and phospholipid levels were increased by treatment with Baygon. The UV spectra of membrane lipids indicated an increased malonaldehyde formation and a diene conjugation band was present at 230-235 nm. The liver weights and liver protein (9000 x g supernatant) content were increased when Baygon was administered at a low dose (Table I). However, a decrease was observed when the animals were treated with a high dose. Baygon at a low dose has a slight effect on the protein content and liver enzymes. However, high doses resulted in a decrease in drug-metabolizing enzymes. The observed increase and decrease in protein content due to treatment with low and high doses, respectively, of Baygonremains to beexplained. The toxiceffits of Baygon were observea as salivation, fasciculations, etc. (Table II). With a high dose of Baygon (25-30 mgjkg) about 6070% of the animals did not survive. The activities of hepatic drug-meta~l~ing enzymes were decreased by low doses and the decrease was much more s~gni~~~t with high doses. Hoffman et al. 15 have also reported a decrease in drug-metabolizing enzymes during administration of DDT at a high concentration. The life span of methyl carbamate insecticides in the animal body is very short and most of the metabolites are eliminated primarily in the urine 1s. It is very likely that the low doses of Baygon may affect the drug-metabolizing enzyme levels by disorganizing the components involved in the drug-metabolizing system. However, as the dose is increased the animal cannot adapt its metabolism and hence death results.
TABLE I EFFECT OF BAYGON TREATMENT ON LlVER WEIGHTS, RELATIVE LIVER WEIGHTS AND m
X &? sUPI%-
NATANT PROTKIN CONTGNT IN YOUNG GROWING MALE RATSa
Baygon dose
Owikd
Control 5 25
Liver wefght
w?
4.12 f 0.1 4.5 f 0.2& 4.0 fO.lb
a S.E.M. rt mean (5 rats in each b P < 0.05. 0 P-cO.01.
Liver weight X body weight
w
3.85 4.12b 4.7=
group).
9000 x g sitperrwtant protein rrt&tt ._._l_____-.-_
211.4 221.8” 201.9”
Total protrirr trig/liver
liver -.-
871.0 997.9f 807.9b
.-..
TABLE II EFF’K!T OF
BAYGON
T~A~NT
ON DRUG
ENZYMES AND
1.9 f 0.01 1.87 I .47 :i f 0.02t 0.02*
11.1 rt 0.2 16.6 14.4 -+ f 0.28 0.39
LIPID
PEROXIDATION
IN YOUNG
GRDWING
MALE RATSa
Control 2:
14.5 f 0.2 11.7 13.1 4& 0.2’ 0.P
4.7 rf 0.1 5.6 f 0.2’ 4.8 O.le
42.5 -49.0*
8.9 -12.58
a S.E.M. f mean (5 rats in each group). b nmoles formal~hyde f~m~d~rnin~rng protein. c Arnold-hydroxya~t~ilide for~/min/mg protein. d nmo!es malonaldehyde fo~ed~min/mg protein. @P < 0.05. r P‘CO.01. g P< 0.001.
The enzymatic and non-enzymatic lipid pet-oxidations were enhanced during Baygon treatment. Lipid peroxidation in the microsomal membrane leads to a loss of membrane structure which could decrease the levels of electron transport chain involved in hydroxylation of drugs and steroids 17. The observed increase in lipid ~roxidation could be explained by an increase in membrane lipids and phospholipids. The presence of an increased UV spectral band of ma~onaIdehyde formation at 260265 rm and a diene conjugation band at 230-235 nm could indicate in viva lipid peroxidation due to treatment with Baygon. During lipid peroxidation hydroperoxides are believed to be formed by oxidation of the unsaturated fatty acids in the &position of the membrane phospholipitis thereby leading to rapid malonaldehyde formationl*alQ. So far no data are available regarding the role of insecticides in microsomal lipid per-oxidation. Our earlier observations20 indicated an increase in lipid peroxidation, showing that membrane bound lipids are affected due to treatment with insecticides. An increase in insecticide concentration generally leads to an increase in inhibition of substrate metabolism and an increase in lipid peroxide formation. ACKNOWLEDGEMENTS
Thanks are due to the C.S.I.R. for awarding a Junior Research Fellowship to Surinder Makhija. The authors are indebted to Professor R. W. Estabrook, Chairman Department of Biochemistry, Southwestern Medical School, University of Texas, DalIas, Tex., U.S.A., for help and encouragement. We are also thankful to Dr. J. M. Pate1 (presently at the Nationai Institute of Environmentai Health Sciences, Research Triangle Park, NC., U.S.A.), N. R. Galdhar and Suniti .Iava~gekar for their help during the course of this work.
299 REFERENCES 1 J. G. Krishna and J. E. Casida, Fate in rats of the radiocarbon from ten variously lab&d methyl- and dimethylcarbamateC1 insecticide chemicals and their hydrolysis products, J. Agrlc. Food Chem., 14 (1966) 98. 2 R. L. Baron and J. P. Doherty, Metabolism and excretion of an insecticide (6-chloro-3&dimethylphenyl N-methylcarbamate in the rat, J. Agric. Food C/tern., 15 (1967) 830. 3 E. S. Oonnitban and J. E. Casida, Oxidation of methyl and dimethylcarbamate insecticide chemicals by microsomal enzyme and anticholinesterase activity of the metabolites, J. Agric. Food Chem., 33 (1969) 1060. 4 J. Miyamoto, K. Yamamoto and T. Matsumoto, Metabolism of 3&dimethylamino-Nmethy’ carbamate in white rats, Agric. Biol. Gem., 33 (1%9) 1060. 5 H. R. Rao and M. W. Anders, Inhibition of microsomal drug metabolism by anticholinesterase insecticides, Bull. Environ. Contam. Toxicol., 9 (1973) 41.
6 J. T. Stevens, R. E. Stitzel and J. J. McPhillips, Effect of anticholinesterase insecticides on hepatic microsomal metabolism, J. Pharmacof. Exptl. Ther., 181 (1972) 576. 7 A. G. Gornall, C. 3. Bardawill and M. M. David, Determination of serum protein by means of biuret reaction, J. Biol. Chem., 177 (1949) WI. 8 J. B. Schenkman, H. Remmer and R. W. Estabrook, Spectral studies of drug interaction with hepatic microsomal cytochrome, Mol. Pharmacol., 3 (1967) 113. 9 T. Nash, The calorimetric estimation of formaldehyde by means of the Hantzsch reaction, Biochem. J., 55 (1953) 416. 10 J. H. Weisburger and C. M. Goodall, Stearic inhibition of enzyme reaction. Lack of enzyme hydrolysis of 2,4,6_trimethylacetanilide, Life Sci., 7 (1968) 263. 11 L. Ernster and K. Nordenbrand, Microsomal lipid peroxidation, Methods in Enzymol., 10 (1967) 584. 12 H. Zalkin and A. L. Tappet, Studies of the mechanism of vitamin E action, lipid peroxidation in the vitamin E deficient rabbits, Arch. Biochem. Biophys., 88 (1960) 113. 13 F. Bernheim, M. L. C. Bernheim and L. M. Wilbur, The reaction between thiobarbituric acid and the oxidation products of certain lipids, J. Biol. Chem., 174 (1948) 247. I4 J. Folch, M. Lees and G. H. S. Stanley, A simple method for the isolation and purification of total lipids from animal tissues, J. Biol. Chem., 226 (1957) 497. 15 D. G. Hoffman, H. M. Worth, J. L. Emmerson and R. C. Anderson, Stimulation of hepatic drug metabolizing enzymes by DDT, the relationship to liver enlargement and hepatotoxicity in the rat, Toxicol. Appi. Pharmacol., 16 (1970) 171. 16 H. W. Dorough, Metabolism of insecticidal methylcarbamates in animals, J. Agric. Food. Chew, 18 (1970) 1015.
17 E. D. Wills, Lipid peroxide formation in microsomes. Relationship of hydroxylation to lipid peroxide formation, Biocherlt. J., 1I3 (1969) 333. 18 H. E. May and P. B. McCay, Reduced triphosphopyridine nucleotide oxidase catalyzed alteration of membrane phospholipids, I. Nature of the lipid alterations, J. Biol. Chem., 243 (1968) 2288. 19 B, K. Tam and P. B. McCay, Reduced triphosphopyridine nucleotide oxidase catalyzed alteration of membrane phospholipids, .I. Biol. Chem., 245 (1970) 2295. 20 S. J. Makhija and S. S. Pawar, Effect of insecticide metabolism and lipid peroxidation in young growing rats. ItId.J. Biochem. Biophys., (In press).
- Pfinfd in
ALTERATKINS JN HEPATIC DRUG METABOLISM AND LfPID PEROXIDATION DURING ADMlNlSTRATION OF BAYGON, A P CIDE
SURIN~~R J. MAKWUA AND SITARAM S. PAWAR*
(ReceivedJuly 9th. 1974) (Revision receivedSeptember18th, 1974)
8UMMARY
Biochemical studies during low- and high-dose administration of Raygon (a pesticide) to young male rats were performed. It was observed that the activities of drug=me~boIi~ng enzymes were decreased even at a low dose of Baygon and the decrease was much more significant during high-dose injections. Lipid peroxidation was increased with a low dose and the increase was much more pronounced with a high dose. Besides these changes the animals showed physical changes such as salivation, fasciculations, etc. The present of conjugated diene absorption patterns and malona~dehyde formation indicts the in viva Iipid ~roxidation due to Baygon administration. Toxic effects leading to death were noted when the animals were injected with a high dose of the pesticide, Baygon above 25 mg/kg.
INTRODUCTION
Phenyl-substituted methyicarbamates are pesticides of the anticholinesterase type. Several investigators have studied both the in viuo and in vitro metabolism of methyi carbamates in rats l-4, Rao and Anderss and Stevens et at.6 have reported a slight decrease in drug metabolism due to carbaryl in rats and mouse. Baygon being a common pesticide in India, there are several instances in which human beings come across this readily available drug as a means for suicide. The accidental exposure of laymen to this drug is also very common. * To whom all correspondenceshould be addressed. Abbreviation: TPNH, triphosphopyridinenucleotide, reduced form.
Bioch:emical studies during the low and high dose administration of this drug might be useful to establish its role in hepatic drug metabolism. The data regarding this aspect is not available and hence the present communication describes the effect of Baygon on the liver microsomal drug-metabolizing enzyme system and lipid ~roxidation. MATERIALS AND METHODS
C.F. strain young male albino rats initially weighing 55-65 g were obtained from Calcutta. The animals were supplied with rat pellets (Hindustan Lever Ltd., Bombay) and water ad 2%. for 15 days before the experiments began. In the injection experiments, Baygon (a gift from Bayer India Ltd., Bombay) was administered by intraperitoneal injection using corn oil as a carrier. The control animals received a placebo. Injections at a dose level of 5 mg/kg low and 25 mg/kg high were made for two days. During the injection period the animals were supplied food and water ad I& and it was observed that rats from both control and experimental groups consumed the same amounts. The animals were decapitated 24 h after the last injection. The livers were excised, minced with scissors, rinsed with 0.9 % ice-cold saline and homogenized (1: 10 w/v) in 0.25 Nice-cold sucrose with a teflon pestle and glass homogenizer. The homogenates were centrifuged at 9000 x g for 20 min in a refrigerated centrifuge (Remi K-24). The 9000 x g supernatant protein content was estimated according to the Biuret method?. The activities of aminopyrine N-demethylase and acetanilide hydroxylase were assayed according to Schenkman et ~1.8 in a medium containing 50 mM Tris-HCI buffer (PI-I 7.4), 5 m&Z MgCls, 8 mM substrates ~aminopyrine and a~taniiide), 0.2 mg TPNH and 2 mg/ml9000 x g supernatant protein in a final volume of 5 ml. All incubations were carried out in air at 37” in a Dubnoff metabolic shaker and terminated by the addition of 10% ice-cold trichloroacetic acid. The formaldehyde formed using aminopyriue as substrate was estimated according to the procedure of Nashs. The ~-hydroxyace~nilide produced during the oxidation of acetanilide was measured according to the method of Weisburger and Goodallie. TPNH-linked lipid peroxidation was assayed as described by Ernster and Nordenbrandll. Non-enzymatic lipid peroxidation was assayed according to Zalkin and Tappei 13. The malonaldehyde produced was measured according to Betnheim et aLf3.
The liver lipids were extracted according to the method of Folch et al.14, and UV spectra were determined to establish the presence of malonaldehyde at 260-265 nm and diene conjugation band at 230-235 nm. RESULTS AND DiSCUSSKbN
Acute treatment with Baygon elicited a slight but significant increase in liver weights whereas chronic treatment decreased the liver weights slightly. The relative liver weights were slightly increased with low dose, however, a significant increase
297 was observed with high dose. The 9000 x g su~rna~nt protein content was slightly increased with low dose and slightly decreased with higb dose of Baygon. The decrease in aminopyrine N-demethylase activities were 9.7 and 19.7% with low and high dose administration of Baygon, respectively. The decrease in acetanilide hydroxylase activity was only 1.6% with low doses, but 22.3% when the animals were given a high dose. Low doses of Baygon resulted in a significant increase in enzymatic and slight increase in non-enzymatic lipid peroxidation. The increase in lipid peroxidation was more significant with high dose. The liver lipid and phospholipid levels were increased by treatment with Baygon. The UV spectra of membrane lipids indicated an increased malonaldehyde formation and a diene conjugation band was present at 230-235 nm. The liver weights and liver protein (9000 x g supernatant) content were increased when Baygon was administered at a low dose (Table I). However, a decrease was observed when the animals were treated with a high dose. Baygon at a low dose has a slight effect on the protein content and liver enzymes. However, high doses resulted in a decrease in drug-metabolizing enzymes. The observed increase and decrease in protein content due to treatment with low and high doses, respectively, of Baygonremains to beexplained. The toxiceffits of Baygon were observea as salivation, fasciculations, etc. (Table II). With a high dose of Baygon (25-30 mgjkg) about 6070% of the animals did not survive. The activities of hepatic drug-meta~l~ing enzymes were decreased by low doses and the decrease was much more s~gni~~~t with high doses. Hoffman et al. 15 have also reported a decrease in drug-metabolizing enzymes during administration of DDT at a high concentration. The life span of methyl carbamate insecticides in the animal body is very short and most of the metabolites are eliminated primarily in the urine 1s. It is very likely that the low doses of Baygon may affect the drug-metabolizing enzyme levels by disorganizing the components involved in the drug-metabolizing system. However, as the dose is increased the animal cannot adapt its metabolism and hence death results.
TABLE I EFFECT OF BAYGON TREATMENT ON LlVER WEIGHTS, RELATIVE LIVER WEIGHTS AND m
X &? sUPI%-
NATANT PROTKIN CONTGNT IN YOUNG GROWING MALE RATSa
Baygon dose
Owikd
Control 5 25
Liver wefght
w?
4.12 f 0.1 4.5 f 0.2& 4.0 fO.lb
a S.E.M. rt mean (5 rats in each b P < 0.05. 0 P-cO.01.
Liver weight X body weight
w
3.85 4.12b 4.7=
group).
9000 x g sitperrwtant protein rrt&tt ._._l_____-.-_
211.4 221.8” 201.9”
Total protrirr trig/liver
liver -.-
871.0 997.9f 807.9b
.-..
TABLE II EFF’K!T OF
BAYGON
T~A~NT
ON DRUG
ENZYMES AND
1.9 f 0.01 1.87 I .47 :i f 0.02t 0.02*
11.1 rt 0.2 16.6 14.4 -+ f 0.28 0.39
LIPID
PEROXIDATION
IN YOUNG
GRDWING
MALE RATSa
Control 2:
14.5 f 0.2 11.7 13.1 4& 0.2’ 0.P
4.7 rf 0.1 5.6 f 0.2’ 4.8 O.le
42.5 -49.0*
8.9 -12.58
a S.E.M. f mean (5 rats in each group). b nmoles formal~hyde f~m~d~rnin~rng protein. c Arnold-hydroxya~t~ilide for~/min/mg protein. d nmo!es malonaldehyde fo~ed~min/mg protein. @P < 0.05. r P‘CO.01. g P< 0.001.
The enzymatic and non-enzymatic lipid pet-oxidations were enhanced during Baygon treatment. Lipid peroxidation in the microsomal membrane leads to a loss of membrane structure which could decrease the levels of electron transport chain involved in hydroxylation of drugs and steroids 17. The observed increase in lipid ~roxidation could be explained by an increase in membrane lipids and phospholipids. The presence of an increased UV spectral band of ma~onaIdehyde formation at 260265 rm and a diene conjugation band at 230-235 nm could indicate in viva lipid peroxidation due to treatment with Baygon. During lipid peroxidation hydroperoxides are believed to be formed by oxidation of the unsaturated fatty acids in the &position of the membrane phospholipitis thereby leading to rapid malonaldehyde formationl*alQ. So far no data are available regarding the role of insecticides in microsomal lipid per-oxidation. Our earlier observations20 indicated an increase in lipid peroxidation, showing that membrane bound lipids are affected due to treatment with insecticides. An increase in insecticide concentration generally leads to an increase in inhibition of substrate metabolism and an increase in lipid peroxide formation. ACKNOWLEDGEMENTS
Thanks are due to the C.S.I.R. for awarding a Junior Research Fellowship to Surinder Makhija. The authors are indebted to Professor R. W. Estabrook, Chairman Department of Biochemistry, Southwestern Medical School, University of Texas, DalIas, Tex., U.S.A., for help and encouragement. We are also thankful to Dr. J. M. Pate1 (presently at the Nationai Institute of Environmentai Health Sciences, Research Triangle Park, NC., U.S.A.), N. R. Galdhar and Suniti .Iava~gekar for their help during the course of this work.
299 REFERENCES 1 J. G. Krishna and J. E. Casida, Fate in rats of the radiocarbon from ten variously lab&d methyl- and dimethylcarbamateC1 insecticide chemicals and their hydrolysis products, J. Agrlc. Food Chem., 14 (1966) 98. 2 R. L. Baron and J. P. Doherty, Metabolism and excretion of an insecticide (6-chloro-3&dimethylphenyl N-methylcarbamate in the rat, J. Agric. Food C/tern., 15 (1967) 830. 3 E. S. Oonnitban and J. E. Casida, Oxidation of methyl and dimethylcarbamate insecticide chemicals by microsomal enzyme and anticholinesterase activity of the metabolites, J. Agric. Food Chem., 33 (1969) 1060. 4 J. Miyamoto, K. Yamamoto and T. Matsumoto, Metabolism of 3&dimethylamino-Nmethy’ carbamate in white rats, Agric. Biol. Gem., 33 (1%9) 1060. 5 H. R. Rao and M. W. Anders, Inhibition of microsomal drug metabolism by anticholinesterase insecticides, Bull. Environ. Contam. Toxicol., 9 (1973) 41.
6 J. T. Stevens, R. E. Stitzel and J. J. McPhillips, Effect of anticholinesterase insecticides on hepatic microsomal metabolism, J. Pharmacof. Exptl. Ther., 181 (1972) 576. 7 A. G. Gornall, C. 3. Bardawill and M. M. David, Determination of serum protein by means of biuret reaction, J. Biol. Chem., 177 (1949) WI. 8 J. B. Schenkman, H. Remmer and R. W. Estabrook, Spectral studies of drug interaction with hepatic microsomal cytochrome, Mol. Pharmacol., 3 (1967) 113. 9 T. Nash, The calorimetric estimation of formaldehyde by means of the Hantzsch reaction, Biochem. J., 55 (1953) 416. 10 J. H. Weisburger and C. M. Goodall, Stearic inhibition of enzyme reaction. Lack of enzyme hydrolysis of 2,4,6_trimethylacetanilide, Life Sci., 7 (1968) 263. 11 L. Ernster and K. Nordenbrand, Microsomal lipid peroxidation, Methods in Enzymol., 10 (1967) 584. 12 H. Zalkin and A. L. Tappet, Studies of the mechanism of vitamin E action, lipid peroxidation in the vitamin E deficient rabbits, Arch. Biochem. Biophys., 88 (1960) 113. 13 F. Bernheim, M. L. C. Bernheim and L. M. Wilbur, The reaction between thiobarbituric acid and the oxidation products of certain lipids, J. Biol. Chem., 174 (1948) 247. I4 J. Folch, M. Lees and G. H. S. Stanley, A simple method for the isolation and purification of total lipids from animal tissues, J. Biol. Chem., 226 (1957) 497. 15 D. G. Hoffman, H. M. Worth, J. L. Emmerson and R. C. Anderson, Stimulation of hepatic drug metabolizing enzymes by DDT, the relationship to liver enlargement and hepatotoxicity in the rat, Toxicol. Appi. Pharmacol., 16 (1970) 171. 16 H. W. Dorough, Metabolism of insecticidal methylcarbamates in animals, J. Agric. Food. Chew, 18 (1970) 1015.
17 E. D. Wills, Lipid peroxide formation in microsomes. Relationship of hydroxylation to lipid peroxide formation, Biocherlt. J., 1I3 (1969) 333. 18 H. E. May and P. B. McCay, Reduced triphosphopyridine nucleotide oxidase catalyzed alteration of membrane phospholipids, I. Nature of the lipid alterations, J. Biol. Chem., 243 (1968) 2288. 19 B, K. Tam and P. B. McCay, Reduced triphosphopyridine nucleotide oxidase catalyzed alteration of membrane phospholipids, .I. Biol. Chem., 245 (1970) 2295. 20 S. J. Makhija and S. S. Pawar, Effect of insecticide metabolism and lipid peroxidation in young growing rats. ItId.J. Biochem. Biophys., (In press).
