Subchronic Effects of Dieldrin and Phenobarbital on Hepatic DNA Synthesis in Mice and Rats

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1 FUNDAMENTAL AND APPLIED TOXICOLOGY 29, (1996) ARTICLE NO 00 Subhroni Effets of Dieldrin and Phenobarbital on Hepati DNA Synthesis in Mie and KYLE L. KOLAJA,* DONALD E. STEVENSON,! JASON T. JOHNSON,* EARL F. WALBORG, jr.,f AND JAMES E. KLAUNIG* 1 *Division of Toxiology, Department of Pharmaology and Toxiology, Indiana University Shool of Mediine, Indianapolis, Indiana ; and fdermigen In., Smithville, Texas Reeived January 13, 1995; aepted August 21, 1995 Subhroni Effets of Dieldrin and Phenobarbital on Hepati DNA Synthesis in Mie and. KOLAJA, K. L., STEVENSON, D. E., JOHNSON, J. T., WALBORG, E. F., JR., AND KLAUNIG, J. E. (1996). Fundam. Appl. Toxiol. 29, Dieldrin, an organohlorine pestiide, has been shown to be hepatoarinogeni in mie but not rats. Phenobarbital, in ontrast, indues hepati tumors in both mie and rats. Previous studies have shown that aute dietary exposure of rats or mie to either dieldrin or phenobarbital produes several liver hanges, inluding entrilobular hypertrophy, indution of hepati ytohrome P450, and inreased liver weight. The present study examined the subhroni effet of dieldrin (0.1, 1.0, 3.0, 10.0 mg dieldrin/kg diet) and phenobarbital (10, 50, 100, 500 mg phenobarbital/kg diet) on the indution of hepati DNA synthesis and hepatoyte lethality in male B6C3F1 mie and male F344 rats. Eight-week-old animals were treated as above and evaluated for hepati DNA synthesis after 7, 14, 21, 28, and 90 days of ontinual treatment to dieldrin or phenobarbital. Maximal indution of hepati DNA synthesis in mie was seen at the 14-, 21-, and 28-day sampling times. In rats, no signifiant inrease in hepati DNA synthesis or hepatoyte lethality was observed at any dose of dieldrin investigated. Phenobarbital produed a signifiant inrease in hepati DNA synthesis in both rat and mouse liver following 7 days of treatment. The indution of DNA synthesis in rat liver was transient, with the labeling index returning to ontrol levels by 14 days of treatment. In ontrast, mie treated with phenobarbital showed a signifiant inrease in hepati DNA synthesis throughout the treatment. In both mie and rats, dieldrin and phenobarbital indued hepati DNA synthesis seletively in the entrilobular region of the hepati lobule. The lak of an inrease in serum enzymes indiative of hepati damage and the absene of liver histopathology in mie or rats fed dieldrin or phenobarbital indiate that the indution of DNA synthesis was not mediated by a ytolethal, ompensatory hyperplasti response, suggesting a mitogeni mehanism. Therefore, the speies-speifi indution of hepati DNA synthesis by either dieldrin or phenobarbital orrelated with the previously observed speies-speifi indution of hepati aner by these two Compounds. S 1996 Soiety of Toxiology 1 To whom orrespondene and reprint requests should be addressed at Division of Toxiology, Department of Pharmaology and Toxiology, Indiana University Shool of Mediine, 1001 Walnut Street, MRF 003, Indianapolis, IN Fax: (317) Dieldrin, an organohloride pestiide used from 1950 through 1970, has been shown to produe hepati tumors in mie but not in other rodents, dogs, or primates following hroni exposure (Stevenson and Walker, 1969; Wright et al., 1977, 1978; NCI 1978). Aute exposure to dieldrin, however, produed similar hepatotoxi effets in both the mouse and the rat, inluding inreased liver weight, hypertrophy of entrilobular hepatoytes, and inreased mirosomal ativity (Wright et al., 1972). These hepati hanges were seen within 7 days of dieldrin treatment and were reversible after essation of dosing (Wright et al., 1972). The indution of hepati aner in the mouse does not appear to be strain speifi. Feeding dieldrin (10 mg/kg) for 2 years produed a signifiant inrease in liver tumors in C3HeB/ Fe mie (Davis and Fitzhugh, 1962) and CF1 male mie (Thorpe and Walker, 1973). Similarly, dietary treatment of dieldrin (as low as 2.5 mg/kg diet) to B6C3F1 mie indued a dose-dependent inrease in hepatoarinogeniity (NCI, 1978). In ontrast, Carworth rats and F344 rats fed dieldrin at doses up to mg/kg diet for 2 years produe no signifiant inrease in hepatoarinogeniity, mortality, or growth rate (Cleveland, 1966; NCI, 1978). Similarly, no inrease in hepati tumor inidene was observed following the hroni feeding of dieldrin to hamsters (Cabral et al., 1979), dogs (Wright et al., 1972), or monkeys (Wright et al., 1978). While the exat mehanism by whih dieldrin seletively indues hepati tumors in mie is not known, dieldrin does not appear to funtion through genotoxi means (Ashwood- Smith, 1981). Dieldrin was not mutageni in baterial mutagenesis assays using either human or mouse liver mirosomal enzymes for ativation nor in other mammalian mutagenesis test systems (Bidwell et al., 1975; Marshall et al., 1976; Shirasu et ai, 1977; Simmons et al., 1977; Ashwood- Smith, 1981). Based on its nonmutageniity and lak of diret DNA interation, dieldrin an be lassified as a nongenotoxi arinogen. Indution of DNA synthesis and ell proliferation by nongenotoxi arinogens has been suggested as a mehanisti means for the prodution of organ-speifi and speies-speifi arinogenesis by this group of ompounds (Cohen and Ellwein, 1988, 1990; Ames and Gold, 1990; Butterworth /96 $12.00 Copyright 1996 by ihe Soiety of Toxiology. All rights of reprodution in any form reserved. Downloaded from

2 220 KOLAJA ET AL. and Goldsworthy, 1991; Cohen and Ellwein 1991; Klaunig et al., 1991). The resulting indution of DNA synthesis and ell proliferation may either inrease the opportunity for the aquisition of spontaneous mutations during normal repliative DNA synthesis (Cohen and Ellwein, 1991) or allow for the seletive lonal expansion of previously initiated ells in the target organ (tumor promotion) (Shulte-Hermann, 1987; Klaunig, 1993). The indution of ell proliferation may be through ytotoxi/ytolethal mehanisms as in the ase of hloroform (Larson et al., 1993), where the indution of liver nerosis by hloroform is answered by a regenerative, ompensatory hyperplasia to replae the neroti ells. Other hepati nongenotoxi agents appear to funtion through diret mitogeni mehanisms (i.e., phenobarbital) where no ytolethality is produed and the ompound appears to indue diretly DNA synthesis and ell proliferation, resulting in an inreased ell number and organ size (Shulte-Hermann, 1987). Several investigators have proposed a signifiant role for the indution of ell proliferation in nongenotoxi arinogenesis (Ames and Gold, 1990; Cohen and Ellwein, 1991), others have questioned any relationship between these two events (Melnik 1992; Huff, 1993; Melnik and Huff, 1993). The purpose of the present study was to ompare the effet of dieldrin, in a dose-responsive manner, on hepati toxiity and the indution of DNA synthesis in the B6C3F1 mouse (a speies sensitive to dieldrin indued hepatoarinogenesis) and the F344 rat (a speies insensitive to dieldrin indued hepati tumors). In addition, similar experiments were performed with phenobarbital, a nongenotoxi mitogeni hepatoarinogen in both rats and mie. These data will provide important mehanisti information as to the possible mode of ation of dieldrin in the indution of mouse liver tumors and ontribute in the development of a meaningful estimation of human risk to dieldrin exposure. MATERIALS AND METHODS Chemials. Dieldrin was obtained from Shell Oil Company (Houston. TX) and Phenobarbital was purhased from Sigma Chemial Co. (St. Louis. MO). NIH-07 diets ontaining 0.0, 0.1, mg dieldrin/kg diet and , 100, 500 mg phenobarbital/kg diet were formulated by Dyets (Bethlehem, PA). Purity of the ompounds (99 2% for dieldrin and 98.5% for phenobarbital) and verifiation of ompound onentrations in the diet were determined by gas hromatography (Saady and Poklis. 1990). Animals. Pathogen-free 6-week-old male B6C3FI mie and Fisher 344 rats were purhased from Harlan Sprague-Dawley (Indianapolis, IN), housed, and treated at the Indiana University Laboratory Animal Researh Center (Indianapolis, IN; AAALAC aredited). All animals were maintained in aordane to the NIH Guide for the Care and Use of Laboratory Animals. Animals were individually housed in polyarbonate ages with filter tops, orn ob bedding, and provided with water and feed ad libitum. After a 2-week quarantine period, animals were randomly plaed into one of the nine dose groups and maintained on test diets until sampling ( mg dieldrin/kg diet or mg phenobarbital/kg diet or ontrol diet). Five rats and five mie were sampled after or 90 days of treatment from eah dose group. One week prior to sampling. osmoti minipumps (Alza Company, Palo Alto, CA) ontaining [ 3 H]thymidine (65-85 Ci/mmol; 0.5 mci delivered per hour) were subutaneously implanted into the five mie and five rats being sampled from eah dose group for that time. Weekly body weights, food intake, and water intake were measured throughout the study. At sampling, animals were euthanized and terminal body weights were reorded. Serum from eah animal was analyzed for the hepati enzymes ALT (alanine aminotransferase) and AST (aspartamine aminotransferase) as an indiation of ytotoxiity (Larson et ai, 1993). Livers were removed, weighed, and examined grossly. The livers were separated into lobes and eah lobe ut into 2- to 4-mm-thik longitudinal strips, whih were fixed in 10% neutral buffered formalin and proessed for histologi analysis. Paraffin setions (5 /xm) were ut from eah blok (three bloks per animal) and stained with hematoxylin and eosin for histopathologi evaluation. Additional setions were proessed for the autoradiographi detetion of DNA synthesis. Autoradiography and quantifiation of DNA synthesis. Slides were dipped in NTB2 photographi emulsion (Eastman Kodak, Rohester, NY), air dried, and stored at -20 C. After 12 weeks, slides were developed and fixed (Eldridge et al ). The autoradiograms were then stained with hematoxylin and eosin and examined for repliative DNA synthesis. A setion of duodenum from the respetive animal was inluded in eah blok to ensure that administration of tntiated thymidine to eah animal had ourred. Cells undergoing DNA synthesis were quantitated in all treatment groups. DNA synthesis was evaluated both in total liver (panlobular) and by hepati lobular loation (entrilobular or periportal). In both ases 100 fields (a minimum of 1000 hepatoytes) were evaluated for DNA synthesis. In the ase of total (panlobular) ounting, mirosopi fields were hosen at random while for periportal hepatoytes within five ells of the portal vein (periportal) or entral vein (entrilobular) were ounted. Hepatoytes were onsidered mid-zonal if they were greater than five ells from either the entral or the portal vein. The labeling index was determined by dividing the total number of labeled ells (labeled nulei) by the total number of hepatoytes ounted X 100. Statistial analysis. Statistial signifiane of the treated ondition ompared to that of the appropriate (similar sampling time) ontrol was determined by ANOVA with a Dunnetts post ho test (Gad and Weil, 1986). Statistial signifiane was set at p < RESULTS All animals survived until sampling. No statistial signifiant differenes in either body weight gains (Fig. 1), food onsumption (data not shown), or water onsumption (data not shown) were seen between ontrol and treated mie and rats. At the onentrations studied neither dieldrin nor phenobarbital produed detetable liver ytolethality (as measured by inreased serum enzymes (ALT or AST) and histopathology, (data not shown). Relative liver weights (liver/ body weight ratios) were inreased in dieldrin and phenobarbital treated rats and mie (Fig. 2). Mie treated with dieldrin showed an inrease in relative liver weight ompared to untreated mie at all four onentrations of dieldrin (10.0, 3.0, 1.0, and 0.1 mg/kg diet) after 7. 14, and 21 days of exposure (Fig. 2a). At later sampling times, only dieldrin onentrations of 10.0 mg/kg (at 28 and 90 days) and 3.0 mg/kg (at 90 days) produed a signifiant inrease in relative liver weight from ontrol. treated with dieldrin (Fig. 2b) showed an inrease in relative liver weight ompared to that seen in untreated ontrols only at the 7-day sampling time after exposure to 10 mg dieldrin/kg. Following treat- Downloaded from

3 SUBCHRONIC EFFECTS OF D1ELDRIN AND PHENOBARBITAL 35 r Dieldrin treatmen t (days) Dieldrin treatmen t (days) Mie I ''henobarbital treatmen t (days) FIG. 1. Body we.ghts in dieldnn-treated male B6C3F1 mie (a), and male F344 rats (b) and phenobarbital-treated male B6C3F1 mie () and male h344 rats (d). Values represent the mean ± SD of the body weight. Statistial signifiane from appropriate ontrol group (*) was determined by ANOVA followed by a Dunnett's post ho (p < 0.05). Individual treatment groups in (a) and (b) are O, ontrol; V, 0.1 mg dieldrin/kg diet- D 1 0 mg dieldrin/ kg diet;, 3.0 mg dieldrin/kg diet; and, 10.0 mg dieldnn/kg diet. Ind.v.dual treatment groups in () and (d) are O. ontrol; V, 10 mg phenobarbital/ kg diet; V, 50 mg phenobarbital/kg diet;, 100 mg phenobarbital/kg diet; and, 500 mg phenobarbital/kg diet ment with phenobarbital both mie (Fig. 2) and rats (Fig. 2d) displayed an inrease in relative liver weight. In mie, all phenobarbital onentrations studied (10, 50, 100, and 500 mg/kg diet) produed an inrease in relative liver weight ompared to ontrol mie after 7 days of treatment (Fig. 2). Phenobarbital onentrations of 50 mg/kg diet, 100 mg/kg diet, and 500 mg/kg diet produed a signifiant inrease in mouse relative liver weight after 14 days, 14 and 21 days, and 14, 21, 28, and 90 days of treatment, respetively. Phenobarbital treated rats exhibited an inrease in relative liver weight after 7, 14, and 21 days of treatment at 100 mg/kg and 500 mg/kg. At 28 and 90 days of treatment, only the 500 mg/kg dose of phenobarbital produed a signifiant inrease in relative liver weight (Fig. 2d). The effet of phenobarbital and dieldrin on total (panlobular) hepatoyte DNA labeling in mouse and rat liver is shown in Fig. 3. Following treatment with dieldrin, an inrease in the hepatoyte labeling index was seen only in the mouse (Fig. 3a). No hange in the hepatoyte labeling index was observed in the rat at any of the dieldrin doses examined (Fig. 3b). In the mouse, only the dieldrin dose of 10.0 mg/ kg produed a signifiant inrease in labeling at all of the time points examined (Fig. 3a). The hepatoyte labeling index in mie treated with 10 mg dieldrin/kg diet was 1.7-fold Downloaded from

4 KOLAJA ET AL r b Mie * I DC ~zi A i } ', ; ' ; M I '' \ - ; : 7 W, \ ' : Dieldrin treatmenl (days) Dildrin iralment (days) 10 Mie d j S; J FIG. 2. Terminal relative liver weights in dieldrin-treated male B6C3FI mie (a), and male F344 rats (b) and phenobarbital-treated male B6C3F1 mie () and male F344 rats (d). Values represent the mean ± SD of the relative liver weight (liver weight/body weight X 100). Statistial signifiane from appropriate ontrol group (*) was determined by ANOVA followed by a Dunnett's post ho (p < 0.05). Individual treatment groups in (a) and (b) are 0, ontrol; 0.1 mg dieldrin/kg diet; S. 1.0 mg dieldnn/kg diet;. 3.0 mg dieldrin/kg diet, and D, 10.0 mg dieldrin/kg diet. Individual treatment groups in () and (d) are 0, ontrol,. 10 mg phenobarbital/kg diet; S. 50 mg phenobarbital/kg diet;. 100 mg phenobarbital/kg diet; and 500 mg phenobarbital/kg diet. greater than that of ontrol after 7 days of treatment, over 3fold that of ontrol after 14, 21, and 28 days of treatment, and dereased to approximately 2-fold of ontrol after 90 days of exposure. Mie exposed to dieldrin at 3.0 mg/kg after 14 days of treatment also showed an inrease (1.8-fold) in labeling index over ontrol. No inrease in the panlobular labeling index from ontrol was seen in mie at any exposure time following treatment with 0.1 and 1.0 mg/kg dieldrin. Treatment with phenobarbital produed an inrease in the hepatoyte labeling index in both mie (Fig. 3) and rats (Fig. 3d). Phenobarbital (500 and 100 mg/kg diet) indued a signifiant inrease in DNA synthesis in the mouse liver after 7. 14, and Downloaded from 21 days of treatment (4-fold, 2.5-fold, and 2-fold inreases, respetively). Phenobarbital at 500 mg/kg also indued a 1.7fold inrease in DNA synthesis after 28 days of treatment (Fig. 3). In ontrast, in the rat, hepati DNA synthesis was indued 3.0-fold by 100 mg/kg phenobarbital and 4.7-fold by 500 mg/kg after 7 days of treatment. After 14 days of exposure, only the 500 mg/kg dose group remained signifiantly elevated (1.4-fold) (Fig. 3d). In mie and rats, phenobarbital onentrations of 50 and 10 mg/kg did not indue DNA synthesis ompared to that seen in ontrol. DNA synthesis in the portal, mid-zonal, and entrilobular regions was quantitated (Figs. 4, 5, and 6). Lobular differ-

5 SUBCHRONIC EFFECTS OF DIELDRIN AND PHENOBARBITAL 223 Mie b x S 2 io,o f 5 Mie d 20 x a i 1 10 FIG. 3. Total DNA synthesis in dieldrin-treated male B6C3F1 mie (a), and male F344 rats (b) and phenobarbital-treated male B6C3F1 mie () and male F344 rats (d). Values represent the mean ± SD of the hepati DNA synthesis labeling index (labeled nulei/total nulei X 100). Statistial signifiane from appropriate ontrol group (*) was determined by ANOVA followed by a Dunnett's post ho (p < 0.05). Individual treatment groups in (a) and (b) are 0, ontrol;, 0.1 mg dieldrin/kg diet; s, 1.0 mg dieldrin/kg diet;. 3.0 mg dieldrin/kg diet; and D, 10.0 mg dieldnn/kg diet. Individual treatment groups in () and (d) are 0, ontrol;, 10 mg phenobarbital/kg diet; S, 50 mg phenobarbital/kg diet;. 100 mg phenobarbital/kg diet; and D, 500 mg phenobarbital/kg diet. enes in labeling indies were noted. In the rat, dieldrin and phenobarbital indued no signifiant inreases in portal DNA synthesis (Figs. 4b and 4d). In the mouse phenobarbital did indue a signifiant inrease in portal DNA synthesis by 1.4- and 1.1-fold after 7 and 14 days of exposure, respetively. In rats, no inreases in periportal hepatoyte DNA synthesis was seen following phenobarbital treatment. In mid-zonal hepatoyte labeling index, dieldrin produed an inrease of 1.5-fold in 3.0 mg/kg and and a 2.9-fold inrease in 10.0 mg/kg after 14 days exposure (Fig. 5a). After 21 and 28 days of exposure, only 10.0 mg/kg dieldrin inrease midzonal labeling index (2.4- and 2.0-fold inreases, respetively). No inrease in midzonal labeling index was seen in rat hepatoytes following dieldrin treatment (Fig. 5b). Phenobarbital produed a signifiant inrease in midzonal mouse hepatoyte labeling index after 7 and 14 days treatment (100 and 500 mg/kg, respetively) and 21 and 28 days exposure (500 mg/kg). In phenobarbital-treated rats an inrease in mid-zonal labeling index was seen only after 7 days of treatment at 100 mg/kg (2.8-fold) and 500 mg/kg (4.1-fold) (Fig. 5d). Dieldrin and phenobarbital produed the most pronouned effet upon DNA synthesis in the entrilobular region (Fig. 6). Centrilobular labeling indies in dieldrin-treated mie Downloaded from

6 224 KOLAJA ET AL. Mie x 20 o C 15 < 10 % 5 Mie X 20 eo 15 "3 XI a < 10 o a <)0 Phenobarbilal treatment (days) FIG. 4. Periportal DNA synthesis in dieldnn-treated male B6C3F1 mie (a) and male F344 rats (b) and phenobarbital-treated male B6C3F1 mie () and male F344 rats (d). Values represent the mean ± SD of the periportal hepatoyte DNA synthesis labeling index (labeled nulei/total nulei X 100). Statistial signifiane from appropriate ontrol group (*) was determined by ANOVA followed by a Dunnetfs post ho (p < 0.05) Individual treatment groups in (a) and (b) are 0, ontrol:, 0.1 mg dieldrin/kg diet: H, 1.0 mg dieldrin/kg diet;. 3.0 mg dieldrin/kg diet; and D, 10.0 mg dieldrin/ kg diet. Individual treatment groups in () and (d) are 0, ontrol;, 10 mg phenobarbital/kg diet; S, 50 mg phenobarbital/kg diet;. 100 mg phenobarbital/ kg diet; and D, 500 mg phenobarbital/kg diet. were signifiantly elevated in the 3.0 and 10.0 mg/kg treatment groups after 7, 14, 21, and 28 days (Fig. 6a). These inreases ranged from 1.4- to 1.8-fold in 3.0 mg/kg treatment group and 2.2- to 8.1 -fold in the 10.0 mg/kg treatment group. After 90 days of exposure, only 10.0 mg/kg dieldrin-treated mie were signifiantly elevated. Dieldrin doses of 0.1 and 1.0 mg/kg did not indue an inrease in mouse entrilobular DNA synthesis. In dieldrin-treated rats, no inrease in entrilobular labeling indies were observed at any dieldrin dose. (Fig. 6b). In phenobarbital-treated mie, an inrease in entrilobular hepatoyte labeling index was seen after 7 and 14 days (100 and 500 mg/kg) and 21 and 28 days (500 mg/kg) of treatment (Fig. 6). In phenobarbital-treated rats, entrilobular DNA synthesis was signifiantly elevated in the 100 and 500 mg/kg treatment groups after 7 days of treatment. After 14 days, only the 500 mg/kg phenobarbital treatment group indued an inrease in entrilobular DNA synthesis. Both phenobarbital and dieldrin produed an inrease in entrilobular hypertrophy in the livers of rats and mie. In dieldrin-treated mie, signifiant entrilobular hypertrophy was seen at all sampling times in the 10 mg/kg treatment group. Mie treated with 3.0 and 1.0 mg dieldrin/kg diet also exhibited entrilobular hypertrophy but to a lesser extent than that seen with the 10 mg/kg dieldrin dose. Mild hyper- Downloaded from

7 SUBCHRONIC EFFECTS OF DIELDRIN AND PHENOBARBITAL 2 Mie b ^ 20 DC C 3 20 BO IS < g 1 10 Mie FIG 5. M,dzonal DNA synthesis in d.eldrin-treated male B6C3F1 mie (a) and male F344 rats (b) and phenobarbital-treated male B6C3F1 mie () and male F344 rats (d). Values represent the mean ± SD of the mid-zonal hepatoyte DNA synthesis labeling index (labeled nulei/total nulei X 100) Statistial s.gnifiane from appropriate ontrol group (*) was determined by ANOVA followed by a Dunnetfs post ho (p < 0.05) Individual treatment groups in (a) and (b) are 0, ontrol; 0.1 mg dieldrin/kg diet;, 1.0 mg dieldrin/kg diet;, 3.0 mg dieldnn/kg diet, and, 100 mg dieldrin/kg diet Indiv.dual treatment groups in () and (d) are 0, ontrol,. 10 mg phenobarb.tal/kg diet;, 50 mg phenobarbital/kg diet;. 100 mg phenobarbital/kg diet; and D, 500 mg phenobarbital/kg diet. trophy was evident in the livers of rats treated with 10 mg/ kg dieldrin. In both mie and rats treated with phenobarbital, entrilobular hepatoyte hypertrophy was apparent. Animals treated with 500 mg phenobarbital/kg diet displayed hypertrophy at all sampling times. In mie and rats treated with 100 mg phenobarbital/kg diet entrilobular hypertrophy was found but to a lesser extent than that seen at the higher dose. DISCUSSION Dieldrin has been examined for arinogeniity in a number of speies inluding mie, rats, dogs, and primates and has repeatedly been shown to be a seletive hepatoarinogen in the mouse (Stevenson and Walker, 1969; Wright et al., 1977, 1978; NCI, 1978). Dieldrin appears to produe its arinogeniity through nongenotoxi mehanisms, being inative in mutagenesis and genotoxi bioassays, (Bidwell et al., 1975; Marshall et al., 1976; Shirasu et al., 1977; Simmons et al., 1977; Ashwood-Smith, 1981). Many nongenotoxi agents have in ommon the property that enables them to indue seletively DNA synthesis and ell proliferation in their target tissue. This property has led several investigators to suggest that indution of DNA synthesis and ell proliferation by nongenotoxi arinogens may be an im- Downloaded from

8 226 KOLAJA ET AL. so a; Mie b > < I I 20 Mie J3 15 I 3 Phenobarbita! treatment (days) ! 28 go FIG. 6. Centrilobular DNA synthesis in dieldrin-treated male B6C3F1 mie (a) and male F344 rats (b) and phenobarbital-treated male B6C3F1 mie () and male F344 rats (d). Values represent the mean ± SD of the entrilobular hepatoyte DNA synthesis labeling index (labeled nulei/total nulei X 100). Statistial signifiane from appropriate ontrol group (*) was determined by ANOVA followed by a Dunnett's post ho (p < 0 05). Individual treatment groups in (a) and (b) are 0, ontrol;. 0.1 mg dieldrin/kg diet; a, 1.0 mg dieldrin/kg diet;. 3 0 mg dieldrin/kg diet; and D mg dieldrin/ kg diet. Individual treatment groups in () and (d) are 0, ontrol:. 10 mg phenobarbital/kg diet; a. 50 mg phenobarbital/kg diet;. 100 mg phenobarbital/ kg diet; and D, 500 mg phenobarbital/kg diet. portant mehanism through whih these ompounds produe aner (Ames and Gold, 1990; Butterworth, 1990; Cohen and Ellwein, 1990). An inrease in DNA synthesis and ell proliferation may inrease the error suseptibility rate for geneti base pairing (Cohen and Ellwein, 1991). This may result in the formation of initiated ells whih an in time lonally expand through either exogenous or endogenous stimulatory pressures. In ontrast, inreased DNA synthesis following exposure to the nongenotoxi arinogen may allow for the seletive indution of ell proliferation and lonal expansion of already present spontaneously initiated ells in the target tissue (tumor promotion) (Shulte-Hermann, 1987). Alternatively, nongenotoxi arinogens an inhibit apoptosis in preneoplasti lesions, leading to enhaned preneoplasti lesion growth (Shulte-Hermann ex al., 1990; Gerbraht ex al., 1990). While the relative role of ell proliferation in arinogenesis is not ompletely understood, it appears to be a neessary omponent of arinogenesis (Butterworth, 1990; Cohen and Ellwein, 1990). Based on the importane of DNA repliation in the aner proess it has been suggested that the measurement of DNA synthesis and ell proliferation an be used toward the establishment of more meaningful assessment of human risk and the existene of thresholds for nongenotoxi Downloaded from

9 SUBCHRONIC EFFECTS OF DIELDRIN AND PHENOBARBITAL 227 arinogens (Cohen and Ellwein, 1990). In the present study, dieldrin indued hepati DNA synthesis in a dose- and speies-responsive manner. The male F344 rat, previously shown to be refratory to dieldrin hepatoarinogenesis did not show any inrease in hepati DNA synthesis following dieldrin exposure. The male B6C3F1 mouse, in ontrast, exhibited an inrease in hepatoyte DNA synthesis that orrelated with the previously reported seletively hepatoarinogeni ativity of dieldrin in this speies (Wright et al, 1972). Phenobarbital, a nongenotoxi hepati arinogen in both rats and mie, indued hepatoyte DNA synthesis in both speies whih orrelated with the previously observed indution of hepati aner. In the mouse both dieldrin and phenobarbital produed a prolong indution of hepati DNA synthesis, while in the rat liver, phenobarbital only produed a signifiant inrease in DNA repliation following 7 and 14 days of treatment. The indution of DNA synthesis by xenobiotis appears to be through either ytotoxi mehanisms or mitogeni mehanisms (Larson et al, 1993; Butterworth, 1990). Following hemially indued ytotoxiity a ompensatory hyperplasti response to the ell nerosis results in inreased DNA synthesis. Chloroform is an example of a ytotoxi nongenotoxi hepati agent whih indues aute hepati ellular nerosis that in turn results in ompensatory ell proliferation and DNA synthesis (Larson et al, 1993). In ontrast, a number of nongenotoxi ompounds may produe their effet through nonytotoxi, mitogeni mehanisms (Malansky and Williams, 1980). In the present study, both dieldrin and phenobarbital appear to funtion through a mitogeni mehanism sine neither ompound indued liver ell nerosis as evidened by the lak of both inrease serum liver enzymes (ALT and AST) and histopathology. Studies by our group and others have shown a strong orrelation between the speies and target organ speifiity of the indution of DNA synthesis by a nongenotoxi arinogen and the previously reported speies and target organ speifiity arinogeniity of the hemial (Loury et al, 1987; Cunningham et al, 1991; Klaunig et al, 1991; Eldridge et al, 1992; Cunningham et al, 1994). In ontrast, others have reported that inreased ell proliferation aused by nongenotoxi arinogens does not orrelate with arinogeniity (Melnik, 1992; Huff, 1993; Melnik and Huff, 1993). One question of onern is that this study as well as many other similar studies have examined the indution of DNA synthesis and not neessarily the indution of ell proliferation by the nongenotoxi arinogen. It is possible that DNA repliation an our without the onomitant indution of ell proliferation. Indeed, dieldrin has been reported to inrease polyploidization in the mouse liver in proportion to its administered dietary onentration (van Ravenzwaay el al, 1987). The dieldrin-indued inrease in liver size in mie has been attributed to both hypertrophy and hyperplasia while in the rat dieldrin produed only an inreased hepatoyte size. (Wright et al, 1972). Ploidy may therefore be inreased in dieldrin-treated mouse liver, but there still may be an inrease in ell proliferation. The indution of polyploidization therefore may be the result of indution of DNA synthesis without the onomitant ell proliferation. Dieldrin and phenobarbital also produed entrilobular hypertrophy in mie and rats. This observation is onsistent with previous reports of the effet of dieldrin and phenobarbital on the liver and appears to be the result of the indution of smooth endoplasmi retiulum and ytohrome P450 enzymes (Wright et al, 1972). The signifiane of regionspeifi DNA synthesis is not yet understood. Our results demonstrated that both dieldrin and phenobarbital indued ell proliferation predominantly in the entrilobular region of the liver. Other nongenotoxi arinogens suh as peroxisome proliferators have been reported to seletively indue DNA synthesis in the periportal region of the liver (Eaho et al, 1991). Interestingly, peroxisome proliferating agents also indue ell proliferation and lonal expansion of a separate group of initiated preneoplasti hepatoytes than does treatment with phenobarbital (Grasl-Kraupp et al, 1993). Our results indiate that dieldrin indues DNA synthesis seletively in the mouse (not the rat) and appears to be through mitogeni mehanisms rather than the result of ytolethality. Other effets inluding hypertrophy and inreased liver/body weight ratio were dramatially more pronouned in dieldrin-treated mie than in rats. 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