Indi an Journal of Experimental Biology Vol. 38, May 2000, pp . 452-456

Acute hepatotoxicity of DDT: Effect on glucocorticoid receptors and serum transcortin

Khemais Ben Rhouma, Olfa Tebourbi & Mohsen Sakly*

Laboratoire de Physiologie Ani male, Faculte des Sciences de Bizerte. Jarzouna 7021. Tunisia

Received 28 July /999; revised 16 FebruGiy 2000

The hepatotoxic effect of I, I bis (p-chlorophenyl) 2,2,2 tri chloroethane (DDT) treatment for I 0 consecutive days has been examined in Wi star rats. DDT exposure increased rel ative liver weight, dose dependently, with a marked decrease of glycogen content and profound histological changes including cytoplasmic vacuolizat ion, signs of necrosi s and nuclear enl argement. The hepatomegaly induced by DDT (50 and I 00 mg/kg body weight day- I) appeared not to be accompanied by a significant alteration of the hepatic glucocorticoid receptor concentration and affi nity while, serum corticosteroid binding globulin level increased slightly wi th the lower dose of the pesticide. It is concluded that a short-term exposure to DDT did not lead to a status stress and , therefore, the hepatotoxic effect of organoch lorine seemed not to be medi ated by endogenous glucocorticoids.

Organochlorine insecticides such as DDT, endrin and lindane have been used worldwide in agriculture. These compounds cause environmental contaminat­ion, pose numerous toxic effects including damage to ti ssues and organs . In particular, they are very neurotoxic' -', have an endocrine effect4

'5 and disturb

the reproductive functions6-8

. The impact of organohalogen pesticides on liver has been also widely studied in vivo and in vitro9

•10 since these

xenobiotics produce morphological and biochemical lesions in affected ti ssues 11

'12

•

Glucocorticoid hormone action requtres an interaction with soluble cytoplasmic receptors of target ti ssues. Two separate receptors for glucocorticoid have been characterized. Type I (mineralocorticoid receptors) have a high affinity for aldosterone, cortisol and corticosterone and a lower affinity for the synthetic glucocorticoid, dexametha­sone13. In contrast, type II (glucocorticoid receptors) have a high affinity for dexamethasone and a lower affinity for · corticosterone and aldosterone 14

'15

• The existence of gl ucocorticoid receptors in the liver, has been well-documented 16

-18

. Plasma glucocorticoids, for the most part, are bound specifically to the transcortin or corticosteroid binding globulin (CBG) which regulates the free fraction, i.e, the biologically active component of the plasma hormone 19

• In mammal s, this binding plasma protein is produced mainly by hepatocytes 16

•

*Correspondent author: E-mail: Mohscn.sak ly@fsb.rnu.tn

In the present study, effects of DDT on liver histology and glycogen content have been evaluated and possible alteration of hepatic glucocorticoid receptors and plasma CBG levels were examined after a short-term exposure to this pesticide .

Materials and Methods Animals and reagents - Wistar male and female

rats weighing I 00-150g were used. The rats were housed under controlled conditions of temperature (22°C) , with 14 hr light/dark cycle. Food and water were offered ad libitum. [I ,2 (n)-3H] dexamethasone ([

3H] dexamethasone,42 ci/mmol) and [I ,2,6,7 -'H] corticosterone (['H) corticosterone, 84 ci/mmol) were obtained from Amersham-France, non-labe led steroids, DDT (98% pp') and all other chemicals were purchased from Sigma Chemical Co. (S t.Loui s, Missouri, USA). Animals received an ip injection of DDT diluted with corn oil at different doses (10, 50 and I 00 mg/kg body weight) . Control rats were admjnistered the vehicle only.

Measurement of [3H/ dexamethasone specif;c binding-The animals, adrenalectomized 24 hr prior, to strip the binding sites from endogenous corticosteroids, were killed by decapitation and livers were perfused in situ with ice-cold homogeni zat ion buffer containing 1.5mM EDT A, 20mM sodium molybdate, I mM dithiothrei tol in 20mM tris-HCI at pH 7.4 (ref. 20) . After weighing, the livers were minced and homogenized in the same buffer with a Teflon homogenizer and centrifuged at I 00,000 g for 45 min at 4°C. The supernatant cytosol was then used

BEN RHOUMA et al.: ACUTE HEPATOTOXICITY OF DDT 453

for receptor analysis. The final protein concentration was 1.6 mg/ml cytosol. Duplicate 200 111 aliquots of cytosol were incubated for 2 hr at 4°C with increasing concentrations of ['H] dexamethasone (3-33 nM) in absence or presence of I 000 fold excess of unlabeled dexamethasone to assess non specific binding. -At the end of the incubation, lOO 111 of dextran-coated charcoal (3.75 % charcoal, 0 .375% dextran T40) were added to cytosol to remove free steroid. Samples were shaken on ice for I 0 min and centrifuged (500g for I 0 min at 4°C) . The radioactivity was assayed in Beckman Ready Safe scintillation cocktail and DPM were determined using a Beckman LS 6000 liquid scintillation counter.

The method of Scatchard21 was used to determine the density and affinity of specific dexamethasone binding sites in the liver cytosol. Protein content was determined by the method of Bradford22 using bovine serum albumin (BSA) as a standard.

Measurement of CBG in serum- The animals were decapitated and trunk blood was collected and centrifuged (500 g for lO min at 4 °C) to obtain serum. To deplete serum of endogenous steroids, 30!11 of a dextran-coated charcoal solution were added to I 00 111 aliquots of serum and kept at 4 °C for I 0 min . After centrifugation at I ,200 g the supernatant was diluted I :50 (v/v) in 40 mM sodium phosphate buffer at pH 7.2. Aliquots (200 11!) of diluted serum were incubated for 2 hr at 4°C with a saturated dose of 74 nM of eH] corticosterone23

. Parallel incubations contained a I 00-fold excess of cold steroid to permit correction for non-specific binding. Bound and

1

unbound moieties were separated by charcoal assay and the binding capacity of CBG was expressed in terms of 11g/dl of serum. Radioactivity counting was done as previously described.

Histological analysis-Small pieces of liver were fixed overnight at room temperature by direct immersion in 4% paraformaldehyde in 0 .1 M phosphate buffer, pH 7 .4. The samples were dehydrated with ethanol and toluene and embedded in paraffin wax. Serial sections (5 11m thick) were mounted on gelatin-coated glass slides and stained with haematoxylin and eosin.

Measurement of liver glycogen- Glycogen was determined according to the method of Good24 and expressed in terms of mg g- 1

• Liver (I g) was minced in the presence of 4 ml of 20% trichloroacetic acid

. (TCA) by Ultra-Turax homogenizer and washed with 4 ml of 20% TCA. The homogenate was centrifuged at 500 g for 5 min and the supernatant containing the

glucide was transvased to another tube. To this, alcohol (8 ml, 95%) was added. and the mixture was heated to 60°C for 5 mjn. After centrifugation, the alcoholic supernatant was discarded and 5 m1 of HzO and I ml of HCI (3N) were added to the glycogen precipitate. Glycogen was hydrolyzed to glucose by leaving tubes in boiling water for I hr. Glucose is then measured by the ortho-toluidine method.

Statistical analysis- Data were analyzed using Stat View 512+ software (Abacus Concept, Inc.) . The results were expressed as means± SE and a comparison of two means was made using Student' s t test.

Results DDT treatment for I 0 consecutive days

significantly increased relative liver weight in a dose­dependent manner in both sexes (Fig. 1). This increase reached I 1.2, 26.3 and 48.4% of control value for the male while for the female, it was 17 .0, 20.1 and 40.3% of control value for I 0, 50 and I 00 mg DDT/kg body weight day" 1

, respectively. Histological analysis showed normal liver structure

in control rats (Fig. 2A). In rats receiving I 0 repeated DDT injections of 100 mg/kg body weight day"' , cytoplasmic vacuolization, focal necrosis and nuclear enlargement of hepatocytes were observed (Fig. 2B). In addition, administration of pesticide significantly reduced the liver glycogen content in a dose­dependent fashion. Hepatic glycogen concentration was 42.10 ± 0.56 mg/g in untreated rats and decreased

Day 0 Day 10 10 mg 50 mg 100 mg

Control DDT Fig !-Effect of DDT treatment on liver relative weight. (P< 0.05)

453

454 INDIAN 1 EXP BIOL, MAY 2000

to 20.12±1.12 mg/g and 8.93±0.93 mg/g following 50 and 100 mg/kg DDT treatment, respectively.

In order to investigate if hepatomegaly caused by DDT was accompanied by alteration of the characteristics of glucocorticoid receptor in liver cytosol, studies were carried out to examine the ('H] dexamethasone binding sites by Scatchard analysis. Daily treatment of rats with 50 mg/kg body weight of DDT for 10 days did not significantly modify the density of glucocorticoid receptors compared with control group (179.0±3.2 vs 173.7±4.1 fmollmg

Fig. 2- Hepatic changes caused by DDT administration to rats (magnification x 1000). (A) Control rat liver lobule. (B) DDT-ten single doses of I 00 mglkg body weight day" 1• Hepatocyte necrosis , cytoplasmic vacuolization and nuclear enlargement (arrows) can be noted

protein). In addition, the apparent dissociation constant (Kd) remained unchanged compared to the control value [4.9±0.2 vs 4.5 ±0.4 nM (Fig. 3)].

Since, in mammals, CBG is produced primarily by the liver, it was determined whether DDT hepatoxicity altered serum transcortin levels. Male rats were used in the study to prevent the stimulatory effect of estradiol on the plasma concentration of CBG25

. After treatment with 10 mg of pesticide/kg body weight, the rate of circulating CBG remained relatively consistent [7.73 ± 0.33 vs 6.83 ± 0.50 flg/dl (Fig. 4)] however, with a dose of 50 mg/kg a moderate increase (about 36%) of the plasma concentration of CBG was observed compared to vehicle-injected controls. Paradoxically, using 100 mg/kg body weight of organochlorine resulted in no significant change in the binding capacity of the transcortin .

Discussion The present observations that DDT induced liver

hyperplasia in a dose-dependent manner with no sexual differences, confirms previous data demonstrating liver hypertrophy caused by many organochlorine compounds~·26 . This hepatomegaly may be related to the enhancement of nuclear DNA

h . d h . f . . . . 12 21 2s AI synt es1s an t e nse o m1totJc actiVIty · · . so, since liver is a regenerative tissue, response was due to pesticide cytotoxicity and/or to the induction of hepatic drug-metabolizing enzymes29

. In rodent liver,

'1' C)

40

~ 30

1! :::> 0 .0 s::

:::>

1i 20 :::> 0

00

10

0

0.20

o Control rats Kd=4.9nM Bmax = 179.0 fmol/ mg P

• DDT treated rats Kd =4.5 nM Bmax = 173.7 fmoll mg P

0.30

Bound (nM)

Fig 3- Scat chard plots of CHJ dexamethasone binding to rat liver cytosol from control or treated rats with DDT. Number of glucocorticoid receptor (Bmax) is expressed in fentomoles/mg protein.

BEN RHOUMA et al.: ACUTE HEPATOTOXICITY OF DDT 455

10

8

Ou_~~~~~~~~u

Control 10 50 100 -------DDT (mg/kg)

Fig 4-Effect of different dose of DDT on serum CBG binding capacity in male rats. (P < 0.05). Number of determinations are indicated in parentheses.

cell proliferation probably plays a crucial role in the initiation of carcinogenesis by inducing errors in

I. . 2810 M h. I . I I . rep tcatiOn ·· . oreover, tsto ogtca a teratwns have been well demonstrated in the liver after intoxication by organochlorines such as DDT and polychlorinated biphenyls (PCBs) 11

•12

.

The present results showed that hepatic injury was accompanied by histological changes including cytoplasm vacuolization, degenerative lesions, nuclear enlargement and biochemical dysfunction with a marked decrease of glycogen tissue content. These observations closely correlate previous studies reporting abnormal mitoses, rough endoplasmic reticulum development, ribosome proliferation and alteration of amino-acid metabolism after chronic exposure to pesticides 11

'31

'32

• Lipid peroxidation has also been identified as an !mportan! factor in cellular damage by pest icides33

, apparently caused by the d . f . . 34 35 pro uct10n o reactive oxygen species· ·· .

Receptors for glucocorticoid hormones in the normal liver have been well described 16

•18

• In order to investigate whether the hepatotoxicity induced by DDT was accompanied by the alteration of the number of glucocorticoid receptors, type II receptor concentration in liver cytosol from treated or untreated rats was determined . The results show that DDT treatment for I 0 days with 50 mg/kg body weight day- ' affected neither the eH] dexamethasone specific binding nor the apparent dissociation constant (Kd) of the liver glucocorticoid receptor, significantly. The reduction of binding capacity of glucocorticoid is a well-known phenomenon of negative regulation or "down regulation" of cytoplasmic steroid receptors initiated by the increase

of circulating Iigand36-38 as it occured after activation

of the hypothalamic-pituitary-adrenal axis by a variety of stressors39

. In the present study, exposure to DDT did not affect hepatic glucocorticoid receptor concentration and affinity. Indeed, it did not alter adrenocortical function without an increase in the secretion of corticosteroid hormones, suggesting that the cytotoxicity of DDT acts directly on hepatic cells.

Corticosteroid binding globulin or transcortin, is the major circulating plasma glycoprotein that binds glucocorticoids with high affinity and specificity. Control of the plasma concentration of CBG may have important regulatory functions governing corticoid actions 19

• Because the liver is the major site of CBG biosynthesis 16

'19 and a good correlation

between serum CBG levels and the amount of CBG­mRNA in the liver was described40

, we have, in the present investigation, examined whether DDT treatment changes the rate of transcortin secretion. The results clearly indicate that serum CBG concentration exhibited a slight increase after 10 days of DDT treatment with 50 mg/kg body weight day" while, with a two fold higher dose of pesticide, the transcortin levels were unaffected. This unexpected observation may be explained either by a rapid and transient response of the liver to pharmacological doses of organochlorine or by a cytotoxic effect altering the biosynthesis and/or the liberation of CBG by hepatocytes . The increase of circulating CBG !evels induced by the dose of 50 mg/kg body weight may be related in part to the well-known "estrogen like" action of the DDr' A2

.

Furthermore, the present results demonstrate that the hepatoxicity, induced by a short-term exposure to DDT, is not accompanied by alteration of the binding capacity and affinity of liver glucocorticoid receptor and it has a moderate effect on the serum CBG levels .

Acknowledgement This work was supported by the Tunisian Ministry

of Higher Education. The authors gratefully acknowledge the technical assistance of Mr Bechir Azib.

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