ANNALS O F CLIN IC A L AND LABORATORY S C IE N C E , Vol. 27, No. 3 Copyright © 1997, Institute for Clinical Science, Inc.

Role o f Oxidative Stress in the Mechanism of Dieldrin’s Hepatotoxicity*f

STEPHEN BACHOWSKI, Ph.D.,* KYLE L. KOLAJA, Ph.D.,§

YONG XU, P h .D .J CYNTHIA A. KETCHAM, M .A .i

DONALD E. STEVENSON, Ph.D.J1 EARL F. WALBORG JR., Ph.D.,11

and JAMES E. KLAUNIG, Ph.D.§

tArm strong Laboratory, Wright-Patterson AFB, OH 45433

and§Division o f Toxicology,

Department o f Pharmacology and Toxicology, Indiana University School o f Medicine,

Indianapolis, IN 46202 and

11Dermigen, Inc., Smithville, TX 78957

ABSTRACT

The production of reactive oxygen species (ROS) by toxic chemicals has been implicated in acute and chronic disease states, including cancer. This increase in cellular ROS can lead to a state of oxidative stress. Many compounds selectively induce hepatic tumors in mice but not rats. The mechanism for the induction of hepatic cancer by these compounds and the observed species selectivity of this effect are not known but may be related to the induction of oxidative stress. Dieldrin is one such compound and is used in the present study to characterize the relationship between oxidative stress and the observed selective hepatotoxicity of dieldrin in mice. It was found that dieldrin induced oxidative stress in the mouse but not the rat, and the observed oxidative stress correlated with the induction of DNA S-phase synthesis. This evidence suggests that the induction of oxidative stress may be a mechanism by which dieldrin and other mouse specific compounds selectively induce their hepatic toxic effects in mice.

* Send reprint requests to: Major Stephen Bachowski, Ph.D., OL AL HSC/OET; Building 79; 2856 G Street, Wright-Patterson AFB, OH 45433-7400.

t Parts of this material were presented at the 28th Central Regional Meeting of the American Chemical Society, “Chemistry in the 90’s and Beyond: Interfacing Chemistry and Toxicology,” Dayton, OH, in 1996 and the 1995 Society of Toxicology, Baltimore, MD.

1960091-7370/97/0500-0196 $02.00 © Institute for Clinical Science, Inc.

OXIDATIVE STRESS IN MECHANISM OF DIELDRIN’S HEPATOTOXICITY 197Introduction

Oxidative stress is defined as “a disturbance in the prooxidant-antioxidant balance in favor of the former, leading to potential damage.”1 Oxidative stress has been associated with a variety of disease conditions such as rheuma­toid arthritis, cancer, Parkinson’s disease, reperfusion injury and inflammation.2 The production of reactive oxygen species (ROS) has been proposed as one mechanism by which xenobiotics may produce oxidative stress and induce liver damage and carcinogene­sis.3’4,5’6’7’8’9’10 Hepatocytes live in a balance of free radical production, free radical scavengers (antioxidants) and repair of damage caused by free radicals.

Carcinogenic xenobiotics may produce oxi­dative stress by (1) direct biotransformations to electrophilic or free radical intermediates,11 (2) inducing or altering enzymatic systems within the cell, such as cytochrome P-450, per­oxisomes and mitochondria, which in turn gen­erate free radicals, (3) depleting or inhibiting normal enzymatic and non-enzymatic antioxi­dant systems which scavenge the free radicals and protect the cell, or (4) overburden the repair mechanisms within the cell (figure 1). The resulting perturbation of the antioxidant/

prooxidant balance can then create a state of oxidative stress in the hepatocytes leading to lipid peroxidation, deoxyribonucleic acid (DNA) damage, or protein alteration. This in turn can lead to altered cellular function, such as decreased gap junctional communication, activation of transcription factors (AP-1, NF-kB), changes in intracellular calcium and intracel­lular pH changes or cell death.12’13,14,15'16

8-Hydroxy-2'-deoxyguanosine (OH8dG) formation and malonedialdehyde (MDA) are markers of oxidative stress. The OH8dG is produced by hydroxylation in the C-8 position of deoxyguanosine residues in DNA by the hydroxyl radical.17,18 Its formation in target organs has been associated with carcinogene­sis.17,19 Xu and coworkers20 have shown the basal OH8dG concentrations in various mouse strains to correlate with the susceptibility of these strains to both spontaneous and chemi­cally induced hepatic cancer. Malonedialde­hyde is a marker of membrane lipid peroxida­tion resulting from the interaction of reactive oxygen species (ROS) and cellular m em ­branes.21 It has been im plicated in DNA adduct formation and in the carcinogenesis process.22 The resulting membrane damage can lead to a loss of cellular homeostasis by changing mem brane characteristics. Mem-

O xid ativ e StressCompound X

Oxidative Stress

Lipid Peroxidation TAltered Cellular Homeostasis (Ca++, pH, GJIC)

Oncogene Activation (AP-1, Bcl-2, NF-kB)

DNA Damage

F igure 1. Sources of oxidative stress and their role in hepatotoxicity.

198 BACHOWSKI, KOLAJA, WU, KETCHAM, STEVENSON, WALBORG, AND KLAUNIG

brane damage and dysfunction results in loss of calcium and other ion transport systems as well as possible loss of gap junction intercellu­lar communication.12'13,1415

The cell has several ways to alleviate the effects of oxidative stress either by repairing the damage (damaged nucleotides and lipid peroxidation byproducts) or by directly reduc­ing the pro-oxidative state via enzymatic and non-enzymatic antioxidants. Non-enzymatic and enzymatic antioxidants have been shown to scavenge free radicals and ROS. Vitamin E, an important non-enzymatic membrane bound antioxidant, can scavenge free radicals thereby preserving the integrity of the plasma mem­brane.23 The resulting tocopheryl radical formed from this process has higher stability and does not cause free radical damage.

Dieldrin is a selective hepatocarcinogen in mice but not rats.24,25,26,27 Reasons for this dif­ference are not apparent and have been seen with a number of compounds.28 Perchloroeth- ylene, methylene chloride, chlordane, hepta- chlor, and dieldrin29,30,31 are all carcinogens in the mouse but not the rat. While the exact cause of these differences is as yet not well defined, oxidative stress may play a central role. Recent work by Stevenson and cowork­ers32 has shown dieldrin-induced S-phase syn­thesis in mouse livers to be reversed by increasing the level of Vitamin E in the diet. These data suggest that dieldrin’s tumoroge- nicity may, in fact, be due to an increased level of oxidative stress in the mouse hepatocyte. Furtherm ore, 8-hydroxy-2'-deoxyguanosine levels and malonedialdehyde were increased in dieldrin treated mouse hepatocytes but not rat hepatocytes.33

The mechanism behind this oxidative dam­age has not been thoroughly investigated. Metabolic studies conducted by Baldwin and Robinson30 and Hutson29 stress that differ­ences do exist in the metabolism of dieldrin in these two species; however, these differences are not sufficient to account for the selective hepatocarcinogenic effect in the mouse. Inter­estingly, Wright and coworkers34 showed the mixed function oxygenase system to be induced in both rats and mice, but concluded

that induction in mice did not offer the same degree of protection. Likewise, the inhibition is shown of gap junction intercellular commu­nication in B6C3F1 mouse hepatocytes could be reversed by the addition of vitamin E.33,35 Therefore, it was hypothesized by us that induction of oxidative stress by dieldrin may contribute to dieldrin’s observed hepatic tox­icity and carcinogenicity.

MethodsC h e m ic a l s

1 ,2 ,3 ,4 ,1 0 ,1 0 -H e x a c h lo ro -6 ,7-epoxy- l,4,4a,5,6,7,8,8a-octahydro-exo-l,4-endo-5,8- dim ethanonapthalene (Dieldrin 99 percent pure) was supplied,* collagenase Type D f Boehringer-Mannheim and fetal bovine serum were purchased.:]: All other chemicals were purchased from one concern.§ All chemicals obtained were of the highest purity available. Methanol, high pressure liquid chromatogra­phy (HPLC) grade, and acetonitrile were pur­ch ased e lsew h ere ." T he 8 -h y d ro x y -2 '- deoxyguanosine standard was prepared by the method of Kasai and coworkers,18 purified by HPLC and stored in -20°C.

A n im a ls

Male B6C3F1 mice and F344 rats (7 to 8 wks old) were obtained.^] Mice or rats were housed in polycarbonated cages at 22 ± 2°C, relative humidity of 50 ± 10 percent, and 12 hr light and 12 hr dark cycle. All animals were fed NIH-07 diet and deionized water ad libitum. After a 1 wk quarantine period, animals were randomly placed in one of two groups, a con­trol group fed NIH-07 diet or the dieldrin treated group fed 10.0 mg dieldrin/kg diet, which was mixed with NIH-07 diets and for­

* Shell Oil Co, Houston, TX.f Indianapolis, IN.$ Hyclone, Logan, UT.§ Sigma Chemical Co., St. Louis, MO.11 Fisher Scientific Co., Fair Lawn, NJ. ì[ Harlan Sprague Dawley Inc., Indianapolis, IN.

OXIDATIVE STRESS IN MECHANISM OF DIELDRIN’S HEPATOTOXICITY 199

mulated into pellets by a commercial sup­plier.** Five animals in each group were euthanized by carbon dioxide and exangui- nated on days 7, 14, 28, 90, and 300 based on in vivo DNA synthesis studies conducted by Stevenson and coworkers.32 The remainder were sacrificed on day 540. At sampling time, mice and rats were weighed, and urine and b lood w ere co llec ted . T he livers w ere removed, rinsed in phosphate buffer saline (PBS), blotted, and weighed. The livers were sectioned into 1 to 2 mm strips. Alternate strips were either snap frozen in liquid nitro­gen or fixed in formalin for 48 hours.

ROS D e t e r m in a t io n in H e p a t o c y t e s

Livers were perfused using an ethylene gly­col tetraacetic acid (EGTA) solution followed by collagenase Type D solution as previously described.36,37 Hepatocytes were isolated, and cell viability was determined by trypan blue exclusion. The isolated cells with 87 to 95 per­cent viability were plated at a density of 33,000 cells/cm2 cultured in Dulbecco’s Modified Eagle’s Medium Nutrient Mixture (DMEM/ F12) media supplemented with 5 percent fetal bovine serum, insulin solution (5 |i,g/ml), gen- tamycin sulfate (25 |xg/ml), and dexametha- sone (0.8 |xg/ml) in a 5 percent carbon dioxide incubator at 37°C and 95 percent humidity. After a 2 hour attachment period, the cells were loaded with 10 mM salicylic acid in the supplemented DMEM/F-12 media for 1 hour.

The loaded cells were washed twice with a Dulbecco’s phosphate buffer saline solution with glucose (DPBS/G), pH 7.2, and exposed to different doses of dieldrin in 500 jxl of DPBS/G for an additional hour. The 100 mM dieldrin was prepared in dimethyl sulfoxide (DMSO) and diluted before each assay. The concentration of DM SO in m edium was 0.05%. At the termination of incubation, 500 jjlI of a 1 0 percent trichloroacetic acid solution were added to the cells. After 15 minutes on

ice, cells were removed from the centrifuge, and the su p e rn a tan t was used for 2,3- dihydroxybenzoic acid (2,3-DHBA) and sali­cylic acid analysis in HPLC.

ROS D e t e r m in a t io n in M ic r o s o m e

The method of Burke and Mayer38 was used to isolate hepatic microsomes. After determi­nation of protein content by the Coomassie Brilliant Blue protein assay,* cytochrome P-450 activity of the microsomes was deter­mined in a fluorescence spectrophotometerf using the method of Burke.39 The aromatic hydroxylation assay was performed according to the method of Floyd,40 except that 1 mM salicylic acid was used as the spin trap agent. The reaction system consisted of 0.5 mg microsome protein, 50 |xl of 5 mM salicylic acid, 10 |xl of 2 mM nicotinamide-adenine- d inu cleo tid e ph o sp h a te [red uced form ] (NADPH), 5 units of glucose-6-phosphate dehydrogenase (G-6-PD) and 100 |xl of 5 mM glucose-6-phosphate (G-6-P).

After adding different doses of dieldrin, final reaction volume was adjusted to 250 |xl with a sucrose-TKM buffer. The final concen­tration of DMSO in the reaction mixture was 0.05%. Reaction was initiated by addition of NADPH and incubated in 37°C water bath for 30 minutes. Reaction was stopped by adding an equal volume (250 (xl) of 10 percent trichlo­roacetic acid. The supernatant from the cen­trifuged sample (12,000 x g for 10 minutes at 4°C) was immediately analyzed on HPLC.

H P L C A nalysis c o n d it io n s f o r A r o m a t ic H y d r o x y l a tio n

The HPLC system consists of a Waters^ 600E pump with a Waters 700 Satellite WISP autoinjector controlled by a Waters Millen­nium 2010 software package using an IBM 486 computer. A series of two (for cells) or three

** Dyets, Inc., Bethlehem, PA.* Bio-Rad Protein Assay kit.t Perkin Elmer 650-10S. t Waters, 510.

2 0 0 BACHOWSKI, KOLAJA, WU, KETCHAM, STEVENSON, WALBORG, AND KLAUNIG

(for microsomes) Waters Nova-Pak C18 col­umns, 4.8 x 100 mm in a Radial-Pak cartridge guarded by a Nova-Pak C18 Guard-Pak Insert were eluted with a 30 mM sodium citrate solu­tion (pH to 4.65 for hepatocytes and pH 4.65 (for hepatocytes) or pH 4.8 (for microsomes) at a flow rate of 1.3 ml/min. 2,3 Dihydroxyben- zoic acid was detected on a CC-5/LC-4C Ame- perometer Detector from BAS System set at 0.1 jxA range, +800 mV potential and 0.1 filter. Salicylic acid was detected on a Waters 484 Tunable Absorbance Detector set at 296 nm. Standards were freshly made each day in a 5 percent trichloroacetic acid solution.

DNA Sy n t h e s is

One week prior to sacrifice, osmotic mini- pum ps§ containing 3H thym idine (65-85 Ci/mmole; 0.5 mCi delivered per hour) were subcutaneously implanted into the five mice or five rats from each dose group. Sections were cut from paraffin embedded liver, dipped in NTB2 photographic emulsion," air dried, and stored at -20°C. After 12 weeks, autoradio­grams were developed (Kodak D-19 developer and fixer) and stained with hematoxylin and eosin. Liver sections were examined for repli­cative DNA synthesis according to Eldridge et al.41 A section of duodenum from each animal was also examined to verify administration of tritiated thymidine. Hepatocytes undergoing DNA synthesis were quantitated. One hun­dred fields (a minimum of 1,000 hepatocytes) were examined per animal. The labeling index for each animal was determined (total number of labeled cells (labeled nuclei) divided by the total number of hepatocytes x 100).

8 -H ydroxy-2 ' - D e o x y g u a n o sin e A n alysis

Liver DNA was isolated using a procedure modified from Marmur.42 The OH8dG was analyzed according to the method of Chung

§ Alza Company, Palo Alto, CA.11 Eastman Kodak, Rochester, NY.

and Xu.43 Digested DNA was analyzed by HPLC-EC detector with the same HPLC sys­tem as used previously. Results are expressed as ratio of OH 8dG /dG uo x 105. Urinary OH8dG was also analyzed by a modified method from that of Shigenaga and Ames.44 The creatinine level of urine was determined using a spectrophotometer* and a creatinine kit.f Results were expressed as fmoles OH8dG per mg creatinine. For liver OH8dG analysis, one Waters^ Nova-Pak C18 column, 4 jjl, 8 x 100 mm in a Radial-Pak cartridge guarded by a Nova-Pak C18 Guard-Pak Insert was eluted with a 10 percent aqueous methanol contain­ing 12.5 mM sodium citrate, 25 mM sodium acetate and 10 mM acetic acid (pH 5.1) at 1.0 ml/min flow rate. For urinary OH8dG analysis, a series of 3 columns was gradually eluted from 100 percent buffer A (2 percent aqueous ace- tonitrile:methanol (7:3) in 25 mM potassium phosphate monobasic (pH 6.7), to 30 percent buffer B (60 percent aqueous acetonitrile- :methanol (7:3) in 25 mM potassium phos­phate monobasic, pH 6.7) in a 30 min period and allowed to stand for 10 min at 1.0 ml/min flow rate. The OH8dG was detected on a CC-5/LC-4C Ameperometer Detector from BAS System set at 0.1 |xA range, +600 mV potential and 0.1 filter for OH8dG detection. Deoxyguanosine was detected on a Waters 484 Tunable Absorbance Detector set at 254 nm.

oi-T o c o p h e r o l A nalysis

The method of Arnaud45 was used for analy­sis of vitamin E (a-tocopherol). The same HPLC system was used as noted previously. One ultrasphere ODS C18 column,§ 5 |x, 150 x 4.6 mm was eluted with dichloromethane: acetonitrile:methanol (20:70:10) at 1.0 ml/min flow rate. A Waters 484 Tunable Absorbance Detector set at 291 nm was used for detection of a-tocopherol.

* CUBAS Mira, .t Sigma Diagnostics, St. Louis, MO.t Waters Corporation, Milford, MA.§ Beckman Instruments, Fullerton, CA.

OXIDATIVE STRESS IN MECHANISM OF DIELDRIN’S HEPATOTOXICITY 2 0 1

MDA A nalysis

The MDA content in urine and liver was analyzed according to the method of Bagchi and coworkers46 via 2,4-dinitrophenylhydra- zine (DNPH) conjugation. Urinary MDA was noted as nmoles of MDA per mg of creatinine and liver MDA as nmoles of MDA per g of liver. The same HPLC system was used as described previously. One ultrasphere ODS C18 columns, 5 |x, 150 x 4.6 mm column was eluted with a 49 percent aqueous acetonitrile and 51 percent water mobile phase at a 1.0 ml/min flow rate. The UV detector was set at 330 nm.

Results

E f f e c t o f D ie l d r in o n t h e G e n e r a t io n o f ROS in L iv e r M ic r o s o m eAND HEPATOCYTES

Liver microsomes isolated from 9-week-old B6C3F1 and F344 rats were treated with sali­cylic acid and 25 |xM dieldrin for 30 minutes. The aromatic hydroxylation products formed by ROS reaction with salicylate were then ana­lyzed by HPLC using an EC detector (figure2). A 2-fold increase in 2,3-DHBA production was observed in liver microsomes isolated

Microsomes

Mouse Rat Induced induced Mouse Rat

Hepatocytes

F ig u r e 2. Values are expressed as mean ± S.D. Mice were 6 to 8 weeks of age. Animals were placed on diets of either NIH-07 or NIH-07 supplemented with10 mg/kg dieldrin for 14 days prior to microsome or hepa- tocyte isolation. A 30 minute microsomal reaction period was used for assay. A 60 minute reaction period was used for hepatocytes. Statis­tical analysis was accom ­plished by first using analysis of variation (ANOVA) with P< 0.05 followed by the post hoc Student’s ‘t’ test. * Indi­cates a significance increase when compared to control (P< 0.05). Values are expressed as (2,3-dihydroxybenzoic acid/SA) x 100.

Mouse Rat InducedMouse

InducedRat

2 0 2 BACHOWSKI, KOLAJA, WU, KETCHAM, STEVENSON, WALBORG, AND KLAUNIG

from mice but not rats. Dieldrin alone did not cause formation of 2,3-DHBA in reaction mix­tures when microsomes were not present, indi­cating that dieldrin does not spontaneously produce radical species.

L ikew ise, 2 ,3-D H B A adduct was no t detected in reactions in which microsomes were omitted or in reactions utilizing heat- treated microsomes (boiled 30 minutes in water bath). Hepatocytes loaded with salicylic acid were treated with 25 jxM dieldrin for 60 minutes (figure 2). Freshly isolated hepato­cytes are utilized to insure an active xenobiotic m etabolism system .47 Mouse hepatocytes showed a two-fold increase in 2,3-DHBA for­mation. No increase in 2,3-DHBA production was observed in rat hepatocytes.A n t io x id a n t /P r o -o x id a n t B a l a n c e in C h r o n ic a l l y D ie l d r in T r e a t e d M ic e

Dieldrin produced an increase of hepatic DNA synthesis in mice compared to control

(figure 3). The labeling index appeared to pla­teau after 14 days of exposure (10 mg/kg = 10.7 percent) and returned to the level noted at 7 days by day 90. By 300 days, the hepatic DNA synthesis had again risen (10 mg/kg = 19.6 per­cent). At all time points, the 10 mg dieldrin/kg diet produced a significant increase above con­trols in mice. A significant decrease in hepatic a-tocopherol was observed compared to con­trol group (figure 4). This observation was con­tinuous throughout the length of the study. Hepatic MDA showed an initial increase in the mouse at days 7 and 14 (figure 5). The decrease in hepatic MDA at later time points may have been a result of the higher concen­trations of hepatic ascorbic acid (data not shown). Urinary MDA in dieldrin treated mice was higher than in the controls at all time points and doses (figure 6). No change of OH8dG levels were observed in mouse livers (data not shown). However, a clear increase in urinary OH8dG was seen at days 14, 28, and

F igure 3. Hepatic DNA S -p h a se s y n th e s i s fo r B6C3F1 mice treated with dieldrin for up to 90 days on either control (0.0 mg diel­drin/kg diet) or 10.0 mg diel­drin/kg diet. Five animals were used per dose group p e r t im e . R e s u lts a re expressed as mean ± S.D. Statistical analysis was per­form ed using analysis of variation (ANOVA) (P <0.05) followed by Student’s ‘t’ test. * Indicates value sig­nificantly different from that of control.

Days on Diet

OXIDATIVE STRESS IN MECHANISM OF DIELDRIN’S HEPATOTOXICITY 2 0 3

7

©3tf) _

•2 5

5cE(0

54

w 0) ~ ô 13

3

1

10 100

F i g u r e 4. H e p a t ic a-tocopherol concentrations for B6C3F1 mice treated with dieldrin for up to 90 days on either control (0.0 mg dieldrin/kg diet) or 10.0 mg dieldrin/kg diet. Five ani­mals were used per dose group per time. Results are expressed as mean ± S.D. Statistical analysis was per­form ed using analysis of variation (ANOVA) (P < 0.05) followed by student “t” test. * Indicates value signifi­cantly different from that of control.

1000Days on Diet

540 days (figure 7), which was parallel to the observed increase in DNA synthesis (urine samples were not taken at 300 days).D ie l d r in I n d u c e d H e p a t o c a r c in o g e n e s isAND THE ANTIOXIDANT/PRO-OXIDANTB a l a n c e

At 540 days, all remaining mice were sacri­ficed. A 96 percent (26/27) tumor incidence was observed in dieldrin treated mice, while only a 13 percent (4/31) was observed in con­trol mice. The tumors of control mice were all eosinophilic (0/5) while dieldrin treated mice had an 82 percent (57/70) basophilic tumor population.Discussion

In this study, significantly different ROS sta­tus was observed during dieldrin metabolism between mice and rats. Only mouse liver microsomes and hepatocytes showed a higher

and dose dependent generation of 2,3-DHBA (figure 2). The findings suggest the involve­ment of oxidative stress in species-specific car­cinogenic effects of dieldrin in these animals. This ROS can be generated by enzyme activity in mitochondria, microsomes, and peroxi­somes in cells. The activity of P-450 enzyme in microsomes can be responsible for ROS gen­eration by futile cycling.48 The cytochrome P-450 system has been suggested to be critical to the carcinogenic process.49

L ubet and coworkers50 contended that there is a strong correlation between phéno­barbital type cytochrome P-450 (P-450 2B) inducers and carcinogenesis. Parke and Ioan- nides48 found that aldrin, the parent com­pound of dieldrin, can engage in futile cycling via the cytochrome P450 IIB system. Dieldrin also induced this isoenzyme strongly in the mouse but only slightly in the rat.51 This oxi­dative stress effect is also supported by in creased levels of 8-hydroxy-2 '-deoxy-

204 BACHOWSKI, KOLAJA, WU, KETCHAM, STEVENSON, WALBORG, AND KLAUNIG

F igure 5. Hepatic malon- dialdehyde concentrations for B6C3F1 mice treated with dieldrin for up to 90 days on either control (0.0 mg dieldrin/kg diet) or 10.0 mg dieldrin/kg diet. Five ani­mals were used per dose group per time. Results are expressed as mean ± S.D. Statistical analysis was per­form ed using analysis of variation (ANOVA) (P <0.05) followed by Student’s ‘t ’ test. * Indicates value sig­nificantly different from that o f control.

Days on Diet

guanosine and malondialdehyde in dieldrin treated mouse hepatocytes but not rat hepato- cytes33 and the reduction of DNA S-phase syn­thesis in B6C3F1 mice induced by dieldrin when supplementing the diet with vitamin E, a potent free radical scavenger.32

The increased ROS in cells can trigger many signaling pathways. These include: (1) hypo- m ethylation and OH8dG form ation,52 (2) accumulation of intra-cellular calcium by the alternation of membrane structure and func­tion resulting from lipid and protein oxidative damage,53 (3) protein kinase C (PKC) activa­tion triggered by calcium and protein oxida­tion, and (4) inhibition of gap junction inter­cellular communication (GJIC) regulated by PKC and membrane oxidation.54,55 In rats fed with a choline deficient diet, hypomethylation of specific DNA sequence cytidine cytidine guanosine guanosine (CCGG) sites within sev­eral genes (c-myc, c-fos, c-Ha-ras) that are involved in growth regulation was found fol­lowing the alteration of mRNA levels.56 Intra­

cellular calcium and pro tein kinase C is required for activation of AP-1 (protein) in oxi­dative stress status.57,58 Both AP-1 and nuclear factor (NF)-kB are modulated by oxidative stress59,60 which might result in an increase in DNA synthesis.

In support of this argument, activation of AP-1 has been associated with increasing glu­tathione S-transferase (GST) levels.59 Interest­ingly, dieldrin also has been reported to induce GST,61 suggesting a possible connection between dieldrin’s selective hepatic action in the mouse and AP-1 induction. Recent find­ings of a correlation between the induction of DNA synthesis and hepatocarcinogenecity of dieldrin62 and the inhibition of this induction by supplementation of a-tocopherol in diet32 provide a path to link oxidative stress, DNA synthesis, and hepatocarcinogenesis.

Lastly, loss of cellular homeostasis may lead to a proliferative event. The increase of MDA in the mouse liver on dieldrin diet is indicative of membrane lipid peroxidation. Changes in

OXIDATIVE STRESS IN MECHANISM OF DIELDRIN'S HEPATOTOXICITY 205

FIG U R E 6 . Urinary malon- dialdehyde concentrations for B6C3F1 mice treated with dieldrin for up to 90 days on either control (0.0 mg dieldrin/kg diet) or 10.0 mg dieldrin/kg diet. Five ani­mals were used p er dose group per time. Results are expressed as mean ± S.D. per mg creatinine. Statistical analysis was p e rfo rm e d using analysis of variation (ANOVA) (P < 0.05) fol­lowed by Student’s ‘t’ test. * In d ica te s value s ig n if i­cantly different from that of control.

Days on Diet

cellular membranes can result in a large vari­ety of functional alterations within the cell which may lead to cellular proliferation or tum or prom otion.13,31 Oxidative stress can induce changes in cellular homeostasis, such as alteration in calcium,14,63 intracellular pH ,15 and inhibition of GJIC.64 Release of calcium ions from cellular stores may, in turn, lead to mitotic events such as activation of the calcium dependent PKC.65 Likewise, many tumor- promoting agents exhibit the ability to disrupt and inhibit GJIC.13,66 It has been previously shown that GJIC in B6C3F1 mouse hepato- cytes was inhibited by dieldrin treatm ent whereas the F344 rat hepatocytes were not. Furthermore, this inhibition was reversed by addition of a-tocopherol to the media.

In contrast, rat hepatocytes exhibited no decrease in GJIC following exposure to diel­d rin . R uch and K laun ig64 have shown paraquat-generated oxygen free radicals to inhibit gap junction intercellular communica­

tion. This may suggest that inhibition of GJIC in mouse hepatocytes is a result of lipid per­oxidation following dieldrin treatment.

In this study, oxidative stress paralleled the increase in DNA S-phase synthesis. The induction of DNA synthesis in subchronic studies is a predictive tool to evaluate the car­cinogenicity of xenobiotics.67’68,69 W hen cells are in S-phase, they are more vulnerable to the genotoxic effects of chemicals since the time for DNA repair is limited and the DNA is more exposed.70 Since hepatic OH8dG levels did not change, it appears that most of the oxidative damage was repaired, as evidenced by elevated urinary OH8dG.

An alternative hypothesis could include the damage occurring at a site other than the liver. This stimulation of DNA synthesis prior to for­mation of preneoplastic foci may allow for the fixation of spontaneously occurring mutations or mutations resulting from genotoxic com­pounds. This would explain the tumor occur-

206 BACHOWSKI, KOLAJA, WU, KETCHAM, STEVENSON, WALBORG, AND KLAUNIG

F ig ure 7. U rinary 8- hydroxy-2 '-deoxyguanosine concentrations for B6C3F1 mice treated with dieldrin for up to 90 days on either control (0.0 mg dieldrin/kg diet) or 10.0 mg dieldrin/kg diet. Five animals were used per dose group per time. Results are expressed as mean ± S.D. per mg creati­nine. Statistical analysis was performed using analysis of variation (ANOVA) (P <0.05) followed by Student’s ‘t’ test. * Indicates value sig­nificantly different from that of control.

Days on Diet

rences observed by dieldrin in the absence of an initiating agent. An increase in hepatic MDA in the mouse preceded the observed increase in DNA synthesis. Malonedialdehyde, a byproduct formed during free radical dam­age of polyunsaturated fatty acids (PUFA), has been shown to be mutagenic to bacteria and mammalian cells and carcinogenic to rats.71

Furthermore, MDA is capable of forming adducts with DNA.22 The return of MDA in dieldrin treated mouse livers to control con­centrations at later time points may be due either to improved removal of MDA from the liver or to an increase in the antioxidant bal­ance of the liver. The urinary MDA concen­tration, however, remains relatively constant throughout the course of this study.

A decrease in a - to co p h e ro l was also observed; a-tocopherol has been shown to inhibit PKC activity which is involved in many cellular proliferation pathways.72 F u rth er­more, work by Charpentier and coworkers73

suggests that the higher concentrations of hepatic and serum a-tocopherol resulting from increase dietary intake increases secretion of transformin growth factor beta (TGF-(3), an an tiproliferative agent. T he decrease in hepatic a-tocopherol observed in this study then might be related to the proliferative event. Hepatotoxicity and carcinogenicity can be modulated by the presence or absence of hepatic vitamin E. In this study, a decrease in hepatic a-tocopherol was observed with diel­drin treatment.

Vitamin E has an ambiguous role in the hepatocarcinogenic process. Vitamin E has been shown to reduce the size and number of gamma-glutamyl transpeptidase (GGT) posi­tive hepatic lesions in F344 rats which were placed on a choline-deficient diet.74 Hendrich and coworkers75 showed 500 ppm dietary vita­min E to suppress focal volume of placental glutathione S-transferase positive altered hepatic foci in female F344 rats induced with

OXIDATIVE STRESS IN MECHANISM OF DIELDRIN'S HEPATOTOXICITY 2 0 7

diethylnitrosamine at 3 months; however, by11 months no difference from the control was observed. In humans, a decreased serum vita­min E was associated with a higher risk of can­cer.76 In contrast, increased dietary vitamin E resulted in enhanced ciprofibrate-induced hepatic carcinogenesis in female Sprague- Dawley rats.77 However, vitamin E, when administered topically, was able to lower the incidence of skin papilloma resulting from 7,12-dimethylbenzja]-antracene (DMBA) ini­tiation and 12-0-tetradecanoylphorbol-13- acetate (TPA) treatm ent.78 This may be a result of Vitamin E ’s ability to down-regulate protein kinase C which is specifically activated by TPA rather than vitamin E ’s role as an anti­oxidant.72 Thus, vitamin E appears to have an ambiguous role in the carcinogenic process. In this study, DNA synthesis returned to basal levels at 90 days even though a-tocopherol lev­els remained low, suggesting that a-tocopherol is not the sole m ediator of this prolifera­tive effect.

It is concluded from this study that dieldrin selectively induces oxidative stress in the B6C3F1 mouse as evidenced by increased hepatic MDA and urinary MDA and OH8dG, as well as an alternation of antioxidants. Fur­therm ore, the increased oxidative stress is associated with the induction of DNA S-phase synthesis which might be related to the diel­drin carcinogenic effects in the mouse liver. Future effort is required to correlate this period of induced oxidative stress with the gene transcription regulation.References

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