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TOXICOLOGY

AND APPLIED PHARMACOLOGY

17,406-417(1970)

Induction Diuron,

of Hepatic and Other

Microsomal Substituted

Enzymes by Herban@, Urea Herbicides172

FLORENCE K. KINOSHITA AND KENNETH P. DUBOIS
Toxicity Laboratory, Department of Pharmacology, University of Chicago, Chicago, Illinois 60637 Received August 28,1969

Induction of Hepatic Microsomal Enzymes by Herban@,Diuron, and Other Substituted Urea Herbicides. KINOSHITA, FLORENCE K., and DUBOIS, KENNETH P. (1970). Toxicol. Appl. Pharmacol. 17, 406-417. 1- [5 - (3a,4,5,6,7,7a - hexahydro- 4,7- methanoindanyl)]- 3,3- dimethylurea (Herban@) and 3-(3,4-dichlorophenyl)-1,1-dimethylurea(diuron) were administeredto adult female rats to determinetheir enzyme-inducingcapacity. After determiningthat these compounds havethe ability to increase the activity of hepatic microsomalenzymes,they were fed to male and femaleweanlingrats in the diet at levelsof 100, 250, 500, 1000,and 2000 ppm. Dose-relatedincreases in the activities of three hepatic microsomal enzymeswere observedwith maximal induction occurring during the first 3 weeksof feeding. All induced enzyme activities decreased during the duration of the 13-weekstudy except for 0-demethylaseactivity. A sex differencewasobservedin the response of the animals.Males were more susceptible than female rats to the enzyme-inducingactivity of thesetwo compounds. Other urea compounds werefed to weanlingand adult female ratsfor 1 weekat a dietary level of 1000ppm. It wasfound that all the herbitidal urea derivatives that were used causedinduction of at least one enzymesystem. The ability of various chemical agents to induce the synthesis of hepatic microsomal enzymes responsible for the metabolism of foreign and natural substanceshas been well established (Conney and Burns, 1962; Conney, 1967). Among the chemicals to which the total population may be exposed repeatedly are the agricultural chemicals. Of thesecompounds it is well known that the chlorinated hydrocarbon insecticideshave the ability to induce many of the microsomal enzymes. Previous studieshave shown that DDT (Hart et al., 1963; Kinoshita et al., 1966), chlordane (Hart and Fouts, 1963), methoxychlor (Hart and Fouts, 1965), aldrin (Boyland and Booth, 1962), heptachlor (Fouts, 1963), and toxaphene (Kinoshita et al., 1966) can cause induction of hepatic microsomal enzymes. It has been recognized that the ability of a compound to induce microsomal enzymesis an important consideration in the conduct of toxicity and metabolic studies, but little systematic work has been done in this area on the quantitative aspectsof enzyme induction. ’ This investigation wassupported by a research grant (ES-00109) from the United States Public HealthService. ’ Presented in part at thesixthannual meeting of theSociety of Toxicology,Atlanta, Georgia, March
23-25, 1967. 406

cause dose-related increases in microsomal enzymes permitting determination of the no-effect levels (Kinoshita et. At 30 days of age the animals were transferred to individual cages and placed on either untreated ground Rockland Rat Diet or on the same feed containing various levels of the experimental compounds.3-dimethylurea (Herban@) and 3-(3.1 % of the body weight. . The animals received 10. They were housed in gang cages in an air-conditioned room maintained between 68” and 75°F and were fed Rockland Rat Diet and water ad libitum for 8 days. Essentially the same procedure has been applied by Hoffman et al. In the initial studies with substituted urea compounds. The present communication contains the results of experiments which demonstrated that substituted urea herbicides cause induction of hepatic microsomal enzymes.ENZYME INDUCTION BY UREA HERBICIDES 407 Previous work in this laboratory resulted in the establishment of procedures for measuring dose-response relationships and no-effect levels for enzyme induction by pesticidal chemicals. For studies in which weanling rats were used. (1968) in a quantitative study of the enzyme-inducing activity of cc. Seven other substituted urea herbicides were investigated less extensively. have the ability to induce microsomal enzymes. The adult female rats used in these studies were of the Holtzman strain obtained at 50 days of age.7-methanoindanyl)]-3. ci Herban METHODS Diuron Animals and treatment.7. male and female rats of the Holtzman strain were obtained at 22 days of age. DDT and toxaphene.2 % solution of carboxymethyl cellulose at a concentration that permitted administration of a volume equal to 0.4-dichlorophenyl)-l. and are not members of the chlorinated hydrocarbon class of pesticides.100. 1966).4. were selected for a detailed study of the hepatic microsomal enzyme-inducing capacity and for determination of the no-effect dietary level for this process.l-dimethylurea (diuron).5. They were maintained in gang cages for 3 days. Thus feeding studies have shown that low dietary levels of the chlorinated hydrocarbon insecticides. The compounds were suspended in a 0..6. Two members of this class of agricultural chemicals. Parnon).30. At 53 days of age they were transferred to individual cages and placed on experimental diets or were injected with the test compound and placed in cages each holding four animals.7ahexahydro-4. l-[5-(3a. groups of three adult female rats (53 days old) were given Herban or diuron by the oral route. al. Herban and diuron have the following chemical structures: Cl N-C ? t- N’ CH. ‘CH.cr-bis(p-chlorophenyl)-3pyridinemethanol (EL-241 . which may exist as residues on food crops. or 200 mg/kg/day of the compounds for 5 days and were sacrificed on the sixth day. The present investigation was part of a program initiated to ascertain whether agricultural chemicals.

or 300 mg/kg/day of Herban or 10. On the sixth day the activity of the phosphorothioate detoxification system and 0-demethylase were measured. phenylurea. Mixing was done with a mechanical mixer (McClellan Batch Mixer).N.500. 1. N. which was measured spectrophotometrically.(p-chlorophenyl) -N’. l.l-dimethylurea (Tenoran@). At the end of 1. and I-(chloro-2-norbornyl)-3. Twelve rats of each sex were placed on each dietary level. The lower doses of diuron caused .3-dimethylurea (Hercules 7175). Statistical analyses were done using the t test of Student. The urea derivatives were dissolved in cornoil.N’. N-(4bromo-3-chlorophenyl)-N’methoxy-N’-methylurea (C-6313).100. 3.3-dimethylurea. Enzyme assays. or 13 weeks three male and three female rats from each treated and untreated group were sacrificed for enzyme assays.250. The animals were placed on the diets at the age of 30 or 53 days in groups of 3 and sacrificed at 37 or 60 days of age.1000.and fed to theanimalsat a levelof 1000ppm. RESULTS Induction of Hepatic Microsomal Diuron Enzymes by the Oral Administration of Herban and The initial experiment which demonstrated the enzyme-inducing activity of Herban and diuron consisted of oral administration of various doses to adult female rats. The increase in activity of this enzyme ranged from 172% to 312 % of normal in the animals treated with Herban. Another sample of liver weighing about 1 g was used for measuring N-demethylase activity by the method of La Du et al. where it may be seen that a dose-related increase in the enzyme activity of the EPN detoxification system occurred with both compounds.dimethyl . EPN (0-p-nitrophenyl phenylphosphonothioate) was used as the substrate for phosphorothioate detoxification.mixedwiththediet. The results of these measurements are shown in Table 1.a.phenylurea (fenuron).408 KINOSHITA AND DUBOIS In subsequentstudies inwhich Herban and diuron were fed in the diet. and p-nitroanisole was used as the substrate for 0-demethylase. 100. (1955) using aminopyrine as the substrate. or 300 mg/kg/day of diuron for 5 days. Groups of 3 animals received 10. Both of these substrates yieldedp-nitrophenol as an end product. Whole liver homogenates were used for each of the assays. male and female rats were started on the experiments at 30 days of age. 30. or 2000 ppm.N’-dimethylurea (monuron). The compounds were dissolved in corn oil (Mazola@) and added to the ground rat diet at levels of 0.l-dimethylurea. l. The details of each of these methods as applied to quantitative measurements of enzyme induction have been described previously (Kinoshita et al. Several other urea derivatives were also fed to weanling and adult female rats for a period of 1 week. 3-[p-(p-chlorophenoxy)phenyl]-l.30..I -methoxyurea (Patoran@). 3-(p-bromophenyl) . 6.cr-trifluoro-m-tolyl)urea (Cotoran@). Animals were sacrificed by decapitation and exsanguination in groups of three for the enzyme assays.l-dimethyl-3-(a. N. 100. 1966). The compounds used for these experiments were urea.1 -methyl. Approximately 1 g of liver was removed for measurement of the activity of a microsomal phosphorothioate detoxification system by the method of Neal and DuBois (1965) and measurement of 0-demethylase activity by the procedure of Netter and Seidel (1964).

100.7 f 0.2 f 0. or 300 mg/kg caused increases in activity to 131x. * Numberof animals.25’ 3. respectively. but the two highest doses caused increases to 144 % and 192 % of control activity.8 f 0.1 f 0. 1 shows the effects of feeding Herban at various dietary levels on the EPN detoxification system of the male rat.8 zt 1.3Sd Avg. both compounds were fed to young male and female rats at various dietary levels for a period of 13 weeks. 179 %.12d 4.ENZYME INDUCTION BY UREA HERBICIDES 409 no significant changes in the activity of this enzyme system.3 It 0. 254 %. Groups each containing 3 animals were sacrificed at intervals for enzyme assays.38c 6. The dietary levels of Herban and diuron presented in this and subsequent figures include only those levels which caused significant changes (P < 0. Panel A of Fig.4 f 1.05d Diuron aActivity determined asmicrograms of p-nitrophenol formedper 50mgof liver in 60min. c Not significantly differentfrom controls (P > 0. f SE 2.85d 7.* 10 30 100 300 10 30 100 300 6 3 3 3 3 3 3 3 3 Percentof control activity 100 172 180 244 312 116 108 144 192 O-Demethylase” Percentof control activity 100 127 131 179 181 110 138 254 335 Compound Control Herban Avg.31d 17.08’ 2. Induction of EPN Detoxification by Dietary Intake of Herban or Diuron After the demonstration of the enzyme inducing activity of Herban and diuron.6 f 0. dSignificantly differentfrom controls(P10. Herban at daily doses of 30. 1 show the effects of these compounds on the activity of the EPN detoxification system.2f 1. TABLE 1 EFFECT OF ORAL ADMINISTRATION OF HERBAN AND DILJRON ON THE HEPATIC MICROSOMAL ENZYME ACTIVITY OF ADULT FEMALE RATS EPN detoxification” Dose hdkglday for 5 days) No.78’ 13. Diuron at these doses caused an increase in 0-demethylase activity to 138 %.31” 9. and 181% of control.7 i 0.336 5. The two compounds differed quantitatively in their effects on the two enzyme systems.08d 2.08d 6.2 f 0. 0-Demethylase activity was also increased by treatment with Herban and diuron. The data in Fig.05). f SE 5.41d 9.8 f 0.13 4.58 6.05) in activity.05) in the activities of the particular enzyme system and the highest level that caused no significant change (P > 0. respectively.5 f 0.5 zt 0.9 4 0.24d 4. In all cases dose-related changes in enzyme activity were observed. respectively.05). and 335 % of control. Herban caused greater induction of the EPN detoxification system. The lowest level of Herban that caused a minimal change in the activity of the EPN detoxification system after 1 week of feeding .6 f 0.3 f 1.58’ 7.4rt 2. while diuron had a greater effect on 0-demethylase.

and 261 % of control activity.410 KINOSHITA AND DUBOIS was 250 ppm. . At 1000 and 2000 ppm there were significant increases in the activity of the EPN detoxification system which were maintained over the 13-week feeding period. although this was a statistically significant change (panel C. 1) a level of 250 ppm caused only a minimal increase in the activity of the EPN detoxification system. Fig. 243 %. 1. FEMPLES FED ap ow WEEKS 60N DIET 013 6 WEEKS ON DET 13 FIG. The levels of 500. Data obtained on female rats fed various levels of diuron (panel D. In female rats fed Herban (Panel B. 1) indicated that 250 ppm caused no significant change in enzyme activity. The levels of 500. At the 500 ppm level of Herban an increase to 155 % of control activity was observed after 1 week of feeding. and 2000 ppm caused induction of the enzyme activity. Rate and extent of induction of the EPN detoxification female rats fed various dietary levels of Herban and diuron. system in the livers of male and activity decreased to a level not significantly different from control by the sixth week of feeding. 1). respectively. 1000 and 2000 ppm of Herban caused increases in enzyme activity to 197 ‘A.1000. At all dose levels the activity decreased after the first week to a level approaching normal by the sixth week. Fig. but only the increases caused by 1000 and 2000 ppm were maintained over the 13-week period of treatment. An increase in the activity of the EPN detoxification system of male rats fed diuron was seen only at the 2000 ppm level. Fig. in 1 week. but the MALES FEDHEREAN 8.

The increased activity was maintained in the female rat at a more constant level than in the male rat. The levels of 100 and 250 ppm caused minimal changes in enzyme activity. 250. The results of these assays are presented in Fig. and 500 ppm caused only minimal increases in the activity of 0-demethylase in females throughout the 13-week period of treatment. while in the female rat there was a gradual increase in activity at the higher dose levels over the 13-week feeding period. The changes in the activity of 0-demethylase of male rats fed Herban are presented in panel A of Fig. 1 indicate that Herban is a more potent inducer of the EPN detoxification system than is diuron. 250 iG MC0 5 d e 2100 0 0 k # 0 01 3 6 13013 6 13 WEEKS CfU DIET WEEKS ON DlET FIG. 232 %. The pattern of response differed in male and female rats fed Herban. Rate and extent of induction of 0-demethylase in the livers of male and female rats fed various dietary levels of Herban and diuron.1000. at the end of 1 week of feeding.ENZYME INDUCTION BY UREA HERBICIDES 411 The data in Fig. and 2000 ppm of Herban caused increases in 0-demethylase activity in the male rat to 198 %. respectively. 2. Dietary levels . The data obtained using female rats fed Herban are presented in panel B of Fig. Levels of 500. Diuron caused minimal increases in the activity of this enzyme in the male rat. The levels of 100. Although these initial high levels of activity were not maintained. Induction of 0-Demethylase by Dietary Intake of Herban or Diuron The same liver samples used to assay the activity of the EPN detoxification system were employed to ascertain the effects of various levels of Herban and diuron on Odemethylase activity. 2. 2. and 219 % of control. where each point represents an average of measurements on the livers of 3 rats. 2. they remainedsignificantly elevated above control levelsforthedurationof the 13-weekstudy.

412 KINOSHITA AND DUBOIS of 1000 and 2000 ppm caused increases to 181% and 206% of control activity. respectively. after this time there were slow decreases over the remainder of the 13-week feeding period to levels approaching control values. Following this there are decreases to lower plateau levels of activity which are still significantly elevated above control levels. M WEEKS ON DIET in the livers of male and female rats fed various . All levels of diuron used in this study caused significant increases in the 0-demethylase activity of the livers of male and female rats. 3. 2 show that there are initial increases in 0-demethylase activity during the first 3 weeks of feeding either Herban or diuron. All levels of Herban used in these studies caused induction of N-demethylase in both the male and female rats although the pattern of response ti 0 01 3 iN DIET of N-demethylase 13 WEEKS FIG. The data presented in Fig. 3. Rate and extent dietary levels of Herban of induction and dim-on. Induction of N-Demethylase by Various Dietary Levels of Herban and Diuron The results of measurements of N-demethylase activity of the livers of rats fed Herban or diuron are presented in Fig. In females the greatest increases were observed at the end of the first week. and the induced activity was maintained with a slight decrease over the entire feeding period. An exception was the 2000 ppm level. in 1 week. Herban caused induction of 0-demethylase in male and female rats to about the same extent. 2 shows data obtained on male and female rats fed diuron. The lower portion of Fig. In male rats the increases in activity were maintained at a relatively constant level over the 13-week period. where the greatest amount of enzyme induction was observed at the end of the feeding period.

3). However. there were exceptions in that there was a gradual increase in the activity of the EPN detoxification system in female rats fed 1000 . A level of 100 ppm caused significant induction of this enzyme in the male rats. in contrast to the males. 3) also exhibited an increased level of N-demethylase activity. all the herbicidal compounds used caused induction of at least one of the three enzyme systems. The induced activity of the females. The nonherbicidal compounds. males being more susceptible than females. Young rats were more susceptible to the inducing effects of monuron. Cotoran. DISCUSSION The results of the present investigation demonstrated that substituted urea herbicides may be added to the growing list of compounds capable of causing induction of hepatic microsomal enzymes. The greatest amount of enzyme induction was seen at the 2000 ppm feeding level.ENZYME INDUCTION BY UREA HERBICIDES 413 differed in the two sexes. The response was not as great in female rats as in males. 1. such as urea. Tenoran. The potency of substituted urea herbicides as enzyme inducers was considerably less than the potency of DDT and toxaphene (Kinoshita et al. Female rats fed Herban (panel B. Diuron was less effective than Herban as an inducer of N-demethylase (panels C and D. Maximal induction was observed in nearly all cases during the first three weeks of feeding the herbicides. The results of feeding 1000 ppm of each of a number of substituted urea compounds for 1 week to weanling and adult female rats are presented in Table 2. By the end of the sixth week the activity of N-demethylase of males and females fed diuron was not significantly different from controls. Fig.l-dimethylurea. was still evident at the end of the sixth week. which caused an increase to 606% of control activity within the first week of treatment. A sex difference in response to the enzyme-inducing activity of the compounds was noted. C-6313. At the levels employed in this study there was a dose-related response in the amount of enzyme induction caused by these compounds. l. but approached levels found in nontreated rats by the thirteenth week. while adults were more susceptible to diuron. In males (panel A. while only the level of 2000 ppm caused any significant change in the activity of the female rat. but the maximal amount of induction for a particular dietary level occurred at the end of the third week of feeding for all levels except 500 ppm. and phenylurea. The EPN detoxification system and N-demethylase were induced to a greater extent by Herban in adults than in weanlings..3-dimethylurea. With the exception of fenuron and Patoran. Fig. 3) a dose-related response was observed with a maximal increase in 1 week. 1966) using the same quantitative procedures for measuring enzyme induction. Fig. Enzyme Induction Female Rats by Other Substituted Urea Compounds Fed to Weanling and Adult The observation that two substituted urea compounds could induce the activity of hepatic microsomal enzymes stimulated interest in examining other members of this class of chemicals for this effect. and Hercules 7175. By the end of the sixth week the N-demethylase activity of all the male rats fed Herban was not significantly different from that of control animals. did not cause any significant change in the activities of any of the three enzyme systems in adult or young rats.

414 KINOSHITA AND DUBOIS .

0 f 0.1 + 2.4.27 2.0 k 0.l.I-dimethylurea (Tenoran) I-[S-(3a .7methanoindanyl)]-3.7 & 0.6 zk 0.7 * 0.40 5.7 + 0.9 * 0.35 3.38 37 60 37 60 37 60 37 60 3 3 8.2 * 1.96 5.6 & 1.5 It 0.31 10.09 131’ 114c 23.0 f 0.38 3 3 5.52 3.8 z!z0.1 f 0.32 146d 96’ 3 3 4.9 f 0.81 3.75 156* 87’ E 3 m z -z 2 3-(p-Bromophenyl)-l-methyl-l-methoxyurea (Patoran) iV-(4-Bromo-3-chlorophenyl)-N’-methoxyN’-methylurea (C-631 3) N-[p-(p-chlorophenoxy)phenyll1.27 3.6 5 0.5.02 6.46 4.87 143d 246d 3 3 9. H z s $ 3: B E G u i3 .5 zt 0.2 f 0.51 149d 107’ 197d 15od 5.05).0 f 0.1 f 0.1 ?c0.0 & 0.2 f 0.0 5 0.4 + 0.72 5 3 9.3-dimethylurea (Hercules 7175) a Activity determined as micrograms of p-nitrophenol formed per 50 mg of liver in 60 min. 7.0 zt 0.4 & 0.72 6.05). bActivity determined as micrograms of 4-aminoantipyrine formed per 100 mg of liver in 30 min.70 7.1 5 0.0 * 0.78 11.79 2.09 7.19 4.3 zk 0.44 3.19 1866 139” 12 3 12. 6.55 8.9 f 0.7 i 0.a-trifluoro-m-tolyl)urea (Cotoran) 37 60 37 60 136’ 141” 35od 193d 136d 90’ 1886 1454 135d 114= 3.2 f 0.7a-hexahydro-4. dSignificantly differentfrom controls(PsO.2 i 0.cr.0 f 0.53 4.38 3.l-Dimethyl-3-(a.7 * 0.79 3.3-dimethylurea (Herban) I-(Chloro-2-norbornyl)-3.77 6.34 157d 136” 94’ 100’ 1476 loo” 84’ loot 181d 261d 188* 129” 13. c Not significantly differentfrom controls(P > 0.85 8.59 5.92 5.39 2.

assays conducted only at the end of a 13-week or a 2-year feeding period probably would fail to indicate the ability of chemicals to cause enzyme induction during the early phase of the feeding period. Factors influencing the metabolism of drugs in liver microsomes. Sot. and BURNS. In fact. 129-l 42. Delaware (diuron. J. H. (1967). less than 100 ppm of diuron for the male. Rev. R. A.. The results of these studies provide a further indication that prolonged feeding periods are not necessary to measure no-effect levels for enzyme induction. Inc. Sci. A. J. Other substituted ureas also caused enzyme induction in weanling and adult female rats when they were fed for 1 week at 1000 ppm. Exptl. 1. FOUTS. The EPN used in enzyme assays was supplied by E. 31-58. (1963). and Fours. Florida (Cotoran @.... Pharmacol. fenuron. J. E.. Tenoran @. Wilmington. and 250 ppm of diuron in females. Biol. Delaware (Herban 8. Pharmacological implications of microsomal enzyme induction. Inc. A similar response was observed in 0-demethylase activity when male rats were fed 2000 ppm of diuron. I.388-392. Delaware. Wilmington. N. Proc. Med. Pharmacol. I. J. CONNEY. 104(3). between 100 and 250 ppm of Herban for the female. Pharmacol. H. The present study demonstrated that the ability of substituted urea herbicides to induce hepatic microsomal enzymes is not limited to Herban and diuron... A similar pattern was observed in our previous study on DDT and toxaphene especially with N-demethylase. ACKNOWLEDGMENT Chemicals used in the feeding studies were kindly supplied by the following companies: Hercules. Hercules 7175. Ann. Factors influencing drug metabolism. G. The metabolic fate and excretion of drugs.. and for the N-demethylase system less than 100 ppm of Herban for the male. J. it seems important to develop information concerning chemical structures that might be expected to alter detoxification enzymes. 819-1092.. CONNEY. Vero Reach. In view of the large number of environmental and industrial chemicals and drugs to which exposure may occur.416 KINOSHITA AND DUBOIS and 2000 ppm of dim-on. (1962).Patoran Q. du Pont de Nemours &Co. Rev. and l. and 500 ppm of diuron for the female. and monuron). Aduan. Ciba Agrochemical Co. HART. . the no-effect dietary levels of Herban and diuron for induction of hepatic microsomal enzymes appear to be for the EPN detoxification system between 100 and 250 ppm of Herban for males and females.and C-6313). The results of the present study contribute toward that objective by providing information which indicates that substituted ureas of the types used in this study may be suspected of causing enzyme induction. L. 19. Acad. Considering the responses obtained at all the intervals used for measurement of enzyme activity in this study. Effects of acute and chronic DDT administration on hepatic microsomal drug metabolism in the rat. Y. Ann. (1962). (1963). du Pont de Nemours & Co.l-dimethylurea). Wilmington. and BOOTH. for the 0-demethylase system less than 100 ppm of either Herban or diuron for males and females. 2. R. E. Inc. 2000 ppm of diuron in males. When the maximal increase in enzyme activity occurred within the first 3 weeks of the feeding period there was always a decrease toward the normal level during the remainder of the feeding period. 114. REFERENCES BOYLAND. 317-366.

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