Early hepatic changes induced in rats by two ...

Carcinogenesis vol.17 no.3 pp.407^12, 1996

Early hepatic changes induced in rats by two hepatocarcinogenicorganohalogen pesticides: bromopropylate and DDT

Grazyna Kostka, Joanna Kopec-Szle.zak1 andDanuta Palut

Department of Environmental Toxicology, National Institute of Hygiene and'Department of Physiopathology, Institute of Haematology and BloodTransfusion, Warsaw, Poland

The aim of the present studies was to describe the effectof two organohalogen pesticides: DDT and bromopropylate,on early changes in rat liver, proposed in the literature tobe useful endpoints in screening of non-genotoxic hepato-carcinogens and/or liver tumor promoters. We investigatedthe effects on the following endpoints: hepatomegaly, mito-genesis (DNA synthesis, mitotic activity, percentage ofbinuclear cells) and cytochrome CYP2B1 -dependent mono-oxygenase induction. The histological and cytochemicalchanges in the liver were also recorded. Male Wistar ratsreceived bromopropylate in one, three or five daily oraldoses of 125, 250 and 500 mg/kg body wt day"1. DDT wasapplied as one, three and five daily oral doses of 24 mg/kgbody wt day"1 (this dose is close to the mean hepato-carcinogenic dose in male Wistar rats: 34.1 mg/kg bodywt. day"1). In the case of both pesticides the early effectsobserved consisted of hepatomegaly accompanied by anincrease in the p-nitroanisole O-demethylase activity andhepatocyte proliferation. Hepatocyte proliferation was elev-ated during the total experimental period. Vacuolatedcytoplasm and evident focal necrosis may suggest that themaximal increase in hepatocyte proliferation, precedinghepatomegaly, is at least partly related to a regenerativeliver response to pesticides. In addition to the above-mentioned early changes, the present findings provide newevidence for the occurrence of dose-dependent abnormalmitoses (and c-mitoses) in the hepatocytes of the bromopro-pylate and DDT treated rats.

Introduction

Many hepatic cytochrome P-450 inducers of the phenobarbit-one—(CYP2B1,2B2) and clofibrate (CYP4A1)—type (1) havebeen shown to act as rodent non-genotoxic hepatocarcinogensand/or liver tumor promoters (2-4). These enzyme inducersincluding peroxisome proliferators, barbiturates and organichalogen compounds are known to produce liver growth viahypertrophy and hyperplasia (2,5). Since during liver growthno distinct signs of toxicity are detected in this organ, it hasbeen concluded that the hyperplastic episode probably accountsfor the initial liver enlargement. Indeed, the major part of theenzyme-inducing compounds elicit only a transient wave of

•Abbreviations: DDT, l,l-(2,2,2-trichloroethylidene)-bis-{4-chlorobenzene);chlorobenzylate, ethyl 4,4'-dichlorobenzylate; dicofol, 2,2,2-trichloro-1,1 -bis-(4-chlorophenyl)-ethanol; bromopropylate, isopropyl 4,4'-dibromobenzylate;chloropropylate, isopropyl 4,4'-dichlorobenzylate; fenarimol, a-(2-chloro-phenyl)-a-{4-chlorophenyl)-5-pyrimidiriemethanol; nuarimol, a-(2-chloro-phenyl)a-(4-fluorophenyl)-5-pyrimidinemethanol.

© Oxford University Press

hepatocyte proliferation during the first days of exposure (4,5).It is, therefore, unlike that such an early and direct mitogeniceffect may be related to subsequent cancer development.However, several lines of evidence support the hypothesis thatcell proliferation plays a crucial role in different steps ofcarcinogenesis (6). Moreover, hepatomegaly, mitogenesis andenzyme induction are caused by a considerable number ofliver non-genotoxic carcinogens and tumor promoters. There-fore, these early changes in rodent liver have been proposedto be useful early endpoints in screening of non-genotoxiccarcinogens (4,7-11).

The group of phenobarbitone-type inducers includes organo-halogen pesticides, e.g. DDT and its structural analogues (12).DDT is a non-genotoxic rodent carcinogen and a potent livertumor promoter (12,13). Furthermore, it has been classified byIARC (13) as probably being carcinogenic to humans (group2B). Chlorobenzylate, dicofol, bromopropylate, chloropropyl-ate, fenarimol and nuarimol, all of them being structurallyrelated to DDT, are still used in plant protection. Although allof these compounds are devoid of any mutagenic activity (14—17), the first three of them have been shown to induce mouseliver tumors after long-term administration (14,16). Moreover,in two-stage hepatocarcinogenicity models all of these com-pounds are active as potent tumor promoters (12,13). Onlyfenarimol (12) and its close analogue nuarimol (18) appear tobe without effect at promotion step.

The aim of our studies was to describe the early responseof rat liver to short-term exposure to organohalogen pesticides.In our previous paper (19) we have investigated the effectof nuarimol on hepatomegaly, mitogenesis (DNA synthesis,mitotic activity, percentage of binuclear cells) and activity ofp-nitroanisole O-demethylase related to cytochrome CYP2B1;the histopathological changes in the liver were also recorded.To describe further this pattern of the early response of ratliver to short-term exposure to organohalogen pesticides, inthe present studies bromopropylate and DDT (in a dose closeto the mean hepatocarcinogenic dose [13]) were used as modelcompounds.

Materials and methodsChemicalsBromopropylate (97%) and DDT (92.2%) were obtained from the Institute ofIndustrial Organic Chemistry, Warsaw; 6-[3H]thymidine (specific activity 1020MBq mol"1) was from the Institute for Research, Production and Use ofRadioisotopes, Prague, Czech Republic; />-nitroanisole was from Fluka BuschAG, Switzerland

Animals and outline of experimentsMale Wistar rats of our own breed (Pzh:WIS) were used. Prior to theexperiments, the rats were acclimatized for 14 days at controlled temperatureof 22 ± 1°C and relative air humidity of 50 ± 10%, with a 12-h light/darkcycle. Rats were fed a commercial diet free from chlorinated hydrocarbonsas assayed by GLC (sensitivity of the method: 0.1-1.0 ^g/kg diet) and freefrom aflatoxins as determined by TLC (sensitivity of the method: 1 Hg/kgdiet), and filtered tapwater ad libitum. In the experiments with bromopropylate,rats weighing 200-220 g were randomly divided into three groups of 24 ratseach and received (in four subgroups of six rats) bromopropylate in doses of0, 125, 250 and 500 mg/kg body wt. day-' (1/40, 1/20 and 1/10 of LD50),

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G.Kostka, J.Kopei-Szlezak and D.Palut

respectively. In the case of DDT three groups of 12 rats each received (intwo subgroups of six rats) DDT in doses of 0 and 24 mg/kg body wt.day"',respectively; the latter dose is close to the mean hepatocarcinogenic dose ofDDT in male Wistar rats (34.1 mg/kg body wt. day"1). Bromopropylate andDDT were administered orally in an olive oil suspension; controls receivedonly the olive oil vehicle. In all of the groups single doses of bromopropylateand DDT (in control subgroups only the vehicle) were administered five timesat 24-h intervals, between 8.00 a.m. and 9.00 a.m. Body weights were recordedevery day. For estimation of DNA synthesis, [3H]-thymidine (1.2 MBq perrat) was injected i.p into all animals I h before sacrifice.

On days 1, 3 and 5, four groups of six rats receiving doses of 0, 125, 250and 500 mg bromopropylate/kg body wt. day"1 (four subgroups), respectively,as well as three groups of six rats receiving DDT in doses of 0 and 24 mg/kg body wt. day" (two subgroups), respectively, were sacrified by decapitationunder anaesthesia. Livers were immediately excised, rinsed of surface blood,blotted to dryness, weighed and frozen in liquid nitrogen. Representativesamples of liver tissue for biochemical and microscopic analysis were takenfrom the right lobe.

Biochemical analysisDNA synthesis measured according to the earlier described method (19) wasexpressed in d.p.m. mg"1 DNA. DNA content was determined by the methodof Burton (20). p-Nitroanisole O-demethylase activity was measured accordingto Netter et al. (21) by the rate of p-nitrophenol generation by the postmitochon-drial fraction of the liver. Protein concentration was determined by the methodof Lowry et al. (22). The metabolism of /r-mtroanisole was expressed as nmolp-nitrophenol mg"' protein h~'.

Histological and cytochemical analysis

Fragments of liver tissue were embadded in paraffin, and 4—5 \xm serialsections were cut and stained with haematoxylin and eosin; cytochemicalanalysis was performed by Feulgen's method. The number of mitoses andthat of binuclear cells were counted in 2000-3000 hepatocytes and expressedper 1000 hepatocytes.

For statistical treatment a two-tailed Student's r-test was used.

Results

Relative liver weight (RLW)Bromopropylate significantly increased RLW to 123, 137 and146% of control after administrations of three oral dosesi.e. 125, 250 and 500 mg/kg body wt. day"1 . After fiveadministrations of the above doses the RLW remained practic-ally unchanged. DDT given in three or five oral doses of 24mg/kg body wt day"1 also significantly increased RLW but tolesser degree (Table I).

O-Demethylase p-nitroanisole activityTreatment of rats with both pesticides resulted in markedinduction of the hepatic of O-demethylase p-nitroanisole activ-ity (Table II). A single oral dose of bromopropylate (125, 250and 500 mg/kg body wt.) caused a 1.6-fold, 1.7-fold, and 1.5-fold increase in activity of this enzyme, respectively. Afterrepeated administration of bromopropylate p-nitroanisole O-demethylase activity was sustained at high levels. However,there was no correlation between the applied doses of bromop-ropylate and enzyme induction.

In the case of DDT, one, and three or five oral administrationsof 24 mg/kg body wt. day"1 caused a 2.0-fold, 2.7-fold, and2.6-fold increase in enzyme activity, respectively.

DNA synthesisThe effect of bromopropylate and DDT on the rate of [3H]thy-midine utilization for nuclear DNA synthesis is ilustrated inTable m.

In bromopropylate-treated rats, DNA synthesis increasedsignificantly in a dose-dependent manner after single adminis-tration of 125, 250, and 500 mg/kg body wt.; the increase inthymidine incorporation into nuclear DNA was 3.4-fold, 4.3-fold, and 4.7-fold, respectively. After prolonged administrationsof the above doses, DNA synthesis tended to decline, thoughthe differences between the groups receiving two highest doses

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Table I. The effect of bromopropylate(RLW) in male Wistar rats

Compound

ControlBromopropylateBromopropylateBromopropylateControlBromopropylateBromopropylateBromopropylateControlBromopropylateBromopropylateBromopropylateControlDDTControlDDTControlDDT

Dose(mg/kg body wt)

0125250500

0125250500

0125250500

0240

240

24

and DDT on

No. of doses

111133335555113355

relative liver weight

Relative liver weight(g/100 g body wt.)

5.6 ± 0.1°5.4 ± 0.16.0 ± 0.26.0 ± 0.15.2 ± 0.26.4 ± 0.2c

7.1 ± 0.2b

7.6 ± 0.4b

5.1 ± 0.26.7 ± 0.3c

6.8 ± 0.3c

7.6 ± 0.2b

5.2 ± 0.35.5 ± 0.15.2 ± 0.35.8 ± O.ld

5.2 ± 0.36.2 ± 0.3d

•Results are mean ± SEM for groups of six rats."Values significantly different from control, P < 0.001.°Values significantly different from control, P < 0 01.dValues significantly different from control, P < 0.05.

Table II. The effect ofO-demethylase activity

Compound


Dose

bromopropylate and DDT on hepatic p-nitroanisolein male Wistar rats

No. of(mg/kg body wt_) doses

0125250500

0125250500

0125250500

0240

240

24

111133335555113355

O-demethylase p-nitroanisole(nmol p-nitrophenol mg"'protein h"')

7.8 ± 0 1°12.7 ± 1.0"13.2 ± 0.6"11 6 ± 0.4"6.8 ± 0.1

13.0 ± 2.3C

13.8 ± 1.4b

12.5 ± 0.2c

6.0 ± 0.410.4 ± 1.0"11.7 ± 0.8*9.6 ± 0.5c

8.2 ± 0.317.7 ± l.l"9.3 ± 0.8

24.8 ± 1.2b

8.4 ± 0.422.0 ± 0.9b

"Results are mean ± SEM for groups of six rats.bValues significantly different from control, P < 0.001.cValues significantly different from control, P < 0.01.dValues significantly different from control, P < 0.05.

of bromopropylate and control animals were still significant.In the case of DDT, DNA synthesis increased 3.4-fold, 2.3-fold, and 2.0-fold over the control after one, three, and fivedays of dosing.

Mitotic activityFor both bromopropylate and DDT the increase in DNAsynthesis was accompanied by an increase in mitotic activityof hepatocytes, with a peak on the first day after start ofexperiments (Table IV). After repeated administration of thecompounds, the number of mitotic figures tended to decline,though the differences between the groups receiving DDT as

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Hepatic changes induced in rats by bromopropylate and DDT

Table III. The effect of bromopropylate and DDT on the rate of[3H]thymidine utilization for liver DNA synthesis in male Wistar rats

Table V. The effect of bromopropylate and DDT on the number ofbinuclear hepatocytes in the liver of male Wistar rats

Compound Dose(mg/kg body wt.)

No. ofdoses

[3H]thymidine incorporation(d.p.m. mg"1 DNA)


0125250500

0125250500

0125250500

0240

240

24

111133335555113355

2350 ± 145*8074 ± 949b

10152 ± 331b

11084 ± 1161b

2541 ± 1335323 ± H5b

7744 ± 692b

121% ± 529*"3049 ± 2163161 ± 3255952 ± 467b

8239 ± 759*2536 ± 2118554 ± 335b

3649 ± 4438262 ± 469b

3532 ± 3547179 ± 475b

"Results are mean i SEM for groups of six rats.bValues significantly different from control, P < 0.001.

Table IV. The effect of bromopropylate and DDT on mitotic activity inhepatocytes of male Wistar rats

Compound Dose No. of(mg/kg body wt) doses

Mitotic activity(no. of mitoses/1000 hepatocytes)


0125250500

0125250500

0125250500

024

024

024

111133335555113355

3.0 ± 2.0"15.0 ± 2.7b

17.0 ± 3.7b

27.3 ± 5.9b

2.4 ± 1.514.4 ± 2.7b

14.7 ± 3.5b

18.2 ± 2.4b

2.2 ± 1.74.2 ± 2.15.0 ± 2.9b

6.2 ± 2.4b

3.0 ± 1.032.4 ± 7.3b

3.6 ± 0.616.6 ± 5.3b

2.6 ± 1.510.8 ± 2.5b

"Results are mean i SEM for groups of six rats.bVaJues significantly different from control, P < 0.05.

well as two higher doses of bromopropylate and controlanimals were still significant.

As shown in Table V, treatment of rats with a single oraldose of bromopropylate (125, 250 and 500 mg/kg body wt.)produced a significant increase in binucleation. However, afterthree and five administrations this number decreased almostto control. For DDT, as compared with control, a singleadministration increased binucleation, although the differencefrom control was not significant. Prolonged administration ofthis compound (3 and 5 days of dosing) caused sustainedincrease in binuclear hepatocytes.

Histological evaluationHistological analysis showed a normal liver structure in controlrats (Figure 1A). In rats receiving bromopropylate in a single

Compound Dose No. of(mg/kg body wt.) doses

No. of binuclear cells/1000 hepatocytes

ControlBromopropylateBromopropylateBromopropylateControlBromopropylateBromopropylateBromopropylaleControlBromopropylateBromopropylateBromopropylateControlDDTControlDDTControlDDT

0125250500

0125250500

0125250500

0240

240

24

111133335555113355

102 ± 8"123 ± 14b

119 ± 8b

128 ± 17b

101 ± 796 ± 12

115 ± 13105 ± 12105 ± 7112 ± 7113 ± 12113 ± 11101 ± 12121 ± 24104 ± 0.6127 ± I2b

104 ± 15129 ± 10*

"Results are mean ± SEM for groups of six rats.bValues significantly different from control, P < 0.05

dose of 125, 250, and 500 mg/kg body wt. day"1, respectively,dose-dependent cytoplasmic vacuolization in the hepatocytesand focal necrosis became evident. Three daily applications ofthe above doses resulted in more extensive diffuse degenerativelesions with high level of focal necrosis; in addition, uponadministration of both higher doses abnormal mitotic figures(and c-mitoses) were found (Figure 1B). After five administra-tions of all three doses the histological picture was differenti-ated depending on the dose. At both higher doses ofbromopropylate (250 and 500 mg/kg body wt. day"1) cytoplasmvacuolization occurred to a slight degree, but a high level offocal necrosis was present (Figure 1C). The mitotic hepatocytesin the anaphase and metaphase exhibited spindle disturbancesresulting in abnormal mitotic figures (and c-mitoses). Afterfive administrations of the lowest dose of bromopropylate(125 mg/kg body wt.day"1) vacuolization of cytoplasm wasreversible and few small necrotic foci were observed; noabnormal mitoses were present. Upon administration of asingle dose of DDT, the liver sections displayed a highpercentage of vacuolated cytoplasm, and evident inflammatoryinfiltrations suggested hepatocyte necrosis. These changes weremore pronounced after three administrations of the dose, asdistinct signs of necrosis in the central lobule zone and morepronounced cytoplasmic vacuolization were found; in addition,abnormal mitotic figures (and c-mitoses) were observed (FigureID). After five administrations of DDT cytoplasmic vacuoliz-ation remained at the same level and abnormal mitotic figureswere present.

Discussion

The present studies of the early changes in rat liver, producedby short-term treatment with bromopropylate and DDT, showedthat these effects consist of hepatomegaly accompanied by anincrease in the hepatic p-nitroanisole metabolism rate and arise in hepatic cellular hyperplasia. Hyperplasia manifesteditself by an increase in nuclear DNA synthesis and a rise inthe mitotic activity. Hepatocyte proliferation was elevatedduring the experimental period, except for the lower doseof bromopropylate. The increase in hepatocyte proliferation,

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G.Kostka, J.Kopec-Szlfzak and D.Palut

B

Fig. 1. Pathological hepatic changes caused by repeated pesticides administration to rats (H and E , 400 X). (A) Control-fragment of rat liver lobule.(B) Bromopropylate-three single doses of 250 mg/kg body wt. day"1. Cytoplasmic vacuolization, signs of hepatocyte necrosis and abnormal mitotic figures(arrows ). (C) Bromopropylate-five single doses of 250 mg/kg body wt. day"1. Signs of hepatocyte necrosis (arrows). (D) DDT-five single doses of 24 mg/kgbody wt. day"1. Cytoplasmic vacuolization, signs of hepatocyte necrosis and abnormal mitotic figures (c-mitosis) (arrows).

appeared to be at least related to a regenerative liver responseto pesticides, since during liver growth, histological signs ofnecrosis and vacuolated cytoplasm were present. Thus, theseresults may suggest both the direct mitogenic effect leadingto hepatomegaly (via hyperplasia and/or hypertrophy) andcytotoxic/regenerative liver response in bromopropylate andDDT treated rats. It is well established that in the liver, inparticular, cell proliferation plays a crucial role in the initiationof carcinogenesis (6,23,24), either by inducing errors in replica-tion or by converting DNA adducts to mutations before DNArepair can occur. However, the type of hepatic proliferationappears to be a major factor in this phenomenon. A compensat-ory or regenerative cell replication as typically seen after

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partial hepatectomy or necrogenic dose of carbon tetrachloride,but no direct mitogenic effect, is effective in the initiation ofchemical carcinogenesis (23,25-27). Moreover, a recurrentregenerative stimuli as well as continued administration ofdirect mitogens (phenobarbitone, DDT, cyproterone acetate,etc.) expand selective growth of initiated hepatocytes at thepromotion step (6,23,28).

The present results also showed an increase in the binuclearhepatocytes in the liver of treated rats. The return to almostnormal level, after prolonged administration of bromopropyl-ate, may suggest cell division of binucleated hepatocytes. Ithas been shown that during regenerative liver growth and aftermitotic stimulation by tumor promoters the binucleation rate

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Hepatic changes induced in rats by bromopropylate and DDT

is reduced (29-31). However, in our studies DDT inducedsustained increase in binucleation during the experimentalperiod.

Unexpectedly, our histological and cytological analysisshowed the presence of dose-dependent abnormal mitoticfigures (and c-mitoses) in the hepatocytes of the bromopropyl-ate and DDT treated rats. The lowest dose of bromopropylate(125 mg/kg body wt.) did not affect the mitoses and can beregarded as a threshold dose for this effect.

Comparison of our previous studies on nuarimol (19) withthe present experiments performed under analogous conditionsindicate several distinct differences in the hepatic responsebetween nuarimol, on the one hand, and bromopropylate andDDT, on the other. The difference was reflected in the characterand the basal rate of hepatocyte proliferation and in thehistological picture. Nuarimol, regarded as a non-genotoxicagent, produced early changes in the liver i.e. hepatomegaly,direct mitogenic effect and enzyme induction, closely resem-bling those caused by most non-genotoxic hepatocarcinogens/direct liver mitogens (32-35).

From the presented studies it can be concluded that in thecase of two rodent hepatocarcinogens, i.e. bromopropylate andDDT administered in the dose close to mean hepatocarcino-genic one, the increase in hepatocyte proliferation, leading tohepatomegaly was at least partly related to a cytotoxicity andregenerative liver response. At the present stage of our studiesit is impossible to foresee the consequence of abnormalmitotic figures (and c-mitoses) in the hepatocytes of thebromopropylate and DDT treated rats. However, it is wellknown that c-mitotic agents cause numerical chromosomechanges i.e. aneuploidy (36,37).

Further studies are required to evaluate all of these earlychanges as potential indices of hepatocarcinogenic activity.

AcknowledgementThis work was supported by Polish Grant no. KBN 4 4509 92 03.

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Received on May 19, 1995; revised on December 5, 1995; accepted onDecember 8, 1995

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