ANNALS O F CLINICAL AND LABORATORY SCIEN CE, Vol. 16, No. 2 Copyright © 1986, Institute for Clinical Science, Inc.

Hepatic Toxicity of Nickel Chloride in Rats*fELINA DONSKOY, M .D .,i MARK DONSKOY, M .D .,$ FARIPOUR FOROUHAR, M .D .,§ CONCETTINA G. G ILLIES, B.S.,§ ABUBAKR MARZOUK, M .D .,t MARILYN C. REID , B.S.,1, ODETTE ZAHARIA, B .S 4 and F. W ILLIAM SUNDERM AN Jr., M .D.,$||

Departments o f $Laboratory Medicine, §Pathology, and ^Pharmacology,

University o f Connecticut Medical School, Farmington, CT 06032

ABSTRACTEnhanced lipid peroxidation was observed in livers of rats killed 24 hr

after sc Injection of nickel chloride (NiCl2) (750 |xmol per kg), as evidenced by 13-fjld increase of conjugated dienes in microsomal lipids and 4-fold increase of thiobarbituric acid (TBA) chromogens in hepatic cytosol. Histo­logic examination of livers from rats killed one to th ree days after NiCl2 injection (500 |xmol per kg) showed microvesicular fatty metamorphosis, mild hydropic degeneration , and foci of inflammation. M icrovesicular steatosis of hepatocytes was confirm ed by electron microscopy. Dose- related increases of serum aspartate aminotransferase (AST) activity (up to 7-fold vs controls) and alanine aminotransferase (ALT) activity (up to 3-fold vs controls) w ere observed 24 hr after injection of NiCI2 (125 to 750 |xmoI per kg); dim inished serum alkaline phosphatase activity (up to 72 percent reduction vs controls) was seen at NiCl2 dosages from 375 to 750 (xmol per kg. D iethyldithiocarbam ate did not influence the effects of NiCl2 on TBA- chromogens in liver homogenates or on serum AST and ALT activities but acted synergistically with NiCl2 to diminish serum alkaline phosphatase activity and to increase serum bilirubin concentration. This study dem on­strates that p aren teral adm inistration of NiCl2 to rats produces acute hepatic toxicity, with enhanced lipid peroxidation, microvesicular stea­tosis, and increased serum AST and ALT activities.

Introduction recent m onographs.0,17 Numerous inves­tigators have studied the toxic effects of

The toxicity and carcinogenicity of nickel compounds on the kidney, lung, nickel compounds in experim ental ani- heart, and endocrine glands, as well as mals and man have been rev iew ed in th e fe tus, b u t th e h e p a tic toxicity of

nickel com pounds has b een relatively* Supported by U.S. Departm ent of Energy Grant

EV-03140 and National Institute of Environmental Health Sciences Grant ES-01337. rats enhances hepatic lipid peroxidation,

neg lected . S underm an e t al21 show ed that parenteral adm inistration of NiCl2 to

Address reprint requests to F. William Sunder- based upon m easurem ents of thiobarbi- man Tr., Departm ent of Laboratory Medicine, Uni- , . n / T r , * \ i i-versity of Connecticut Health Center, 263 Farming- turlC aCld (TBA) chrom ogens in liver ton Avenue, Farmington, CT 06032. hom ogenates. This study corroborated

1080091-7370/86/0300-0108 $01.50 © Institute for Clinical Science, Inc.

HEPATIC TOXICITY O F NICKEL CHLORIDE 1 0 9

the observation of Faw ade and Pawar7 th a t h e p a tic m icrosom es from N iC l2- treated rats produce increased amounts of TBA-chromogens, when incubated in vitro in the presence of reduced nicotin- am ide-adenine d inucleotide phosphate (N A D PH ) or ascorbate . T he p re se n t paper describes further studies of NiCl2 hepatotoxicity in rats, including (a) cen­trifugal fractionations of TBA-chromo­gens in liver homogenates; (b) m easure­m ents o f co n ju g a ted d ien es in lip ids extracted from hepatic microsomes; (c) histologic examinations of liver by light and electron microscopy; (d) assays of serum am in o tran sfe rase and alkaline phosphatase activities; (e) determ inations of serum bilirubin and nickel concentra­tions; and (f) tests of the effects of dieth- y ld ith io carb am ate , a N i[II]-chela to r. T hese b iochem ical and m orphological studies dem onstrate that a single paren­te ra l in jec tio n of N iC I2 can p ro d u ce acute, dose-related hepatocellular dam ­age in rats.M aterials and M ethods

The te s t substances w ere u ltrap u re n ic k e l c h lo r id e (N iC I2 * 3 H 20 ) and sodium diethyldithiocarbam ate (DDC), recrystallized according to Baselt e t al.1 The experim ental animals w ere 302 male rats of the Fischer-344 strain (body wt. = 190 to 260 g), fed Purina laboratory chow and water ad libitum. Control rats w ere treated with NaCl vehicle solution (140 mmol per L, 0.2 to 0.3 ml per rat), by sc and/or im injections at the same tim e that test rats received injections of NiCl2 (125 to 750 (xiriol per kg, body wt., sc) and/or diethyldithiocarbam ate (1.0 or1.3 mmol per kg, body w t., im).* Food

* For comparison, the acute (two-week) mortality indices for sc administration of NiCl2 to male Fischer rats are as follows: LD5 = 310 |jimol per kg; LD50 = 420 (xmol per kg; LD95 = 560 |xmol per kg; median time between injection and death = 3.4 days (range = 2 to 5 days).22 Acute LD50 values for DDC by ip administration to rats are 6.7 mmol per kg (West and Sunderman27) and 7.2 mmol per kg (Frank et al8).

was rem oved from the cages 17 hr prior to s a c r if ic e . B lo o d sa m p le s w e re obtained by cardiac puncture or aspira­tion from the retro-orbital venous plexus under e th er anesthesia. Liver hom oge­n a te s (one g of l iv e r to fo u r m l of m edium ) w ere p repared w ith a m otor- driven Teflon-glass tissue homogenizer. For m easurem ents of TBA-chromogens, th e hom ogenization m edium was KC1 solution (154 mmol per L, 4°C); for mea- s u re m e n ts o f c o n ju g a te d d ie n e s in microsomal lipids, the hom ogenization m e d iu m was p h o sp h a te -K C l-E D T A solution (KH2P 0 4, 20 mmol/L; N aO H , 15.7 m m ol/L; KC1, 134 m m ol/L; eth- ylenediam ine tetraacetic acid (EDTA), one m m ol p e r L; p H 7.4; 4°C). T he homogenates were filtered through fine- m esh plastic gauze and centrifuged at18.000 g for 15 min at 4°C to sedim ent nuclei, m itochondria, and cell debris; th e su p e rn a te s w ere c e n tr ifu g e d at105.000 g for 60 min at 4°C to sedim ent microsomes.

The following analytical m ethods w ere used: (a) m easu rem en t of hepatic dry weight;21 (b) assay of TBA-chromogens,21 with results expressed as nmol malon- d ia ldehyde (MDA) p e r g (dry w t.) of liver; (c) extraction of microsomal lipids and spectrophotom etric assay of conju­g a ted d ie n e s ;10 (d) m e a su re m e n t of m icrosom al lip id c o n c e n tra tio n s by dichrom ate oxidation;4 (e) kinetic assays of serum asp a rta te am in o tran sfe rase (AST) and a lan in e a m in o tra n sfe rase (ALT) ac tiv itie s3 and serum alkaline phosphatase activity;20 (f) determ ination of serum b ilirub in by th e diazo reac­tion;14 and (g) analysis of serum nickel by electrotherm al atomic absorption spec­trophotom etry with Zeeman background correction.19

F or histologic exam ination by light microscopy, liver was fixed in 10 percent neutral buffered formalin; tissue sections were stained with hematoxylin and eosin and by th e periodie-acid-Schiff (PAS) techn ique (with and w ithout diastase

11 0 DONSKOY, DONSKOY, FOROUHAR, GILLIES, MARZOUK, REID, ZAHARIA, AND SUNDERMAN

treatm ent). For e lec tro n m icroscopy, liver was diced in buffered glutaralde- hyde a t 0°C, p o st-fix ed in b u ffe re d osmium tetroxide, em bedded in plastic, stained with uranyl acetate-lead citrate, and exam ined by use o f a Philips-300 electron microscope at 60 kV.

S tatistical tests (standard deviation , paired and non-paired t tests) w ere p er­form ed according to R osner;16 experi­mental results are given as means ± SD.

ResultsL i p i d P e r o x i d a t i o n

Six pairs of rats w ere killed 24 hr after sc injection of NiCl2 (750 ¡Jimol per kg) or NaCl vehicle solution (controls). Livers of NiCl2-treated rats w ere enlarged and congested, w ith increased w et weights (liver weight = 7.1 ± 1.0 g in NiCl2- treated rats, P < 0.01 versus 5.5 ± 0.8 g in p a i r e d c o n tro ls ) a n d in c r e a s e d liver:body weight ratios (liver weight = 3.2 ± 0.3 p e rcen t of body w eight in NiCl2-treated rats, P < 0.01 versus 2.5

± 0.2 percen t in paired controls). H epat­ic dry weights averaged 34.0 ± 0 .5 p e r­cent of w et weight in NiCl2-treated rats, which did not differ significantly from the corresponding values (33.9 ± 1.0 percent) in paired controls. C onsistent with previous findings,21 the mean con­c e n tra tio n of T B A -ch ro m o g en s was increased 2.3-fold in liver homogenates from N iCl2-trea ted ra ts (figure 1, left panel). As shown in the cen ter panel (fig­ure 1), concentrations of TBA-chromo­gens w ere (a) increased 2-fold in the18,000 g sed im en t, w hich con ta ined nuclei, mitochondria, and cell debris; (b) not significantly increased in the micro­somal pellet; and (c) increased 4-fold in the cytosol (105,000 g supernate). Con­jugated dienes in microsomal lipids were increased 13-fold, com pared to paired controls (figure 1, right panel). Spectro- photom etric scans of microsomal lipids from paired N iCl2-trea ted and control rats are illustrated in figure 2, together with the characteristic difference spec­trum of conjugated dienes (absorption maximum = 233 nm).

s£•<as

r 1̂ Mean ±SD * P < 0.05

ICell debris, nuclei, and mitochondria

hepaticmicro­somes

cytosol(105,000supernate)

Controls NICl2 (750 it mol I kg, 24 hr)

F ig u r e 1. E ffects of NiCl2 (750 |ji.mol per kg, sc, 24 hr before sacrifice) on biochemical indices of hepatic lipid peroxidation. L e ft Panel: C o n cen tra ­tions of TBA-chromogens in liver homogenates from six NiCl2-treated and six control rats. Center Panel: TBA-chromogens in sub- cellular fractions of liver homogenates, obtained (a) by c e n t r i f u g a t i o n a t18.000 g for 15 min (cell debris, nuclei, and mito­chondria), (b) by centrifu­gation at 105,000 g for 60 min (microsomes), and (c) h e p a t i c c y to s o l ( th e105 .000 g su p e rn a te ) . C oncentrations of TBA- chromogens in subcellular f ra c tio n s from th e six

NiCl2-treated rats are expressed as multiples of paired values obtained by simultaneous analyses of 18,000 g sediment, 105,000 g sediment, and 105,000 g supernate from liver homogenates of the six NaCl-treated control rats. Right Panel: Concentrations of conjugated dienes in lipid extracts of hepatic microsomes from six NiCL-treated and six control rats.

HEPATIC TOXICITY OF NICKEL CHLORIDE 111

wave length (nm) wave length (nm)

F ig u r e 2 . Diene conjugation absorption spectra of lipid extracts of hepatic microsomes from a NiCl2- treated rat (750 fxmol per kg, sc, 24 hr before sacri­fice) and a paired control rat. The lipid concentra­tions were 0.5 mg per ml of cyclohexane. Left Panel: Spectrophotom etric curves obtained w ith lipid extracts of hepatic microsomes in the sample cuvette and cyclohexane in the reference cuvette. Right Panel: Difference spectrum obtained by subtracting the absorption curve of hepatic microsomal lipids from a control rat from the corresponding curve of lipids from a paired NiCl2-treated rat.

H is t o p a t h o l o g ic a l F in d in g s

In a se ria l-sac rific e s tu d y , 12 ra ts received sc injection of NiCl2 (500 jxmol per kg) and four rats received sc injec­tion of NaCl vehicle solution (controls). T hree NiCl2-treated rats and one control rat w ere killed at each of the following intervals post-injection; 6, 24, 48, and 72 hr. On light microscopic exam ination, livers from control rats appeared normal. The most notable finding in livers from N iC l2-trea ted rats was m icrovesicular fatty metamorphosis, which was focal at six h r post-in jection and becam e p ro ­gressively more prom inent at 24, 48, and 72 hr (figure 3). O ther morphologic find­ings in livers from N iCl2-trea ted rats included (a) mild hydropic degeneration, (b) variable congestion, and (c) scattered foci of inflammation. There was no evi­dence of hepatic necrosis or cholestasis. Small deposits of PAS-positive, diastase- resistant material w ere noted in Kupffer cells. This PAS-positive material, which m ay re p re s e n t cy to p lasm ic p ro te in s released from damaged hepatocytes, was evident at 24 to 72 hr post-injection in

NiCl2-treated rats bu t was also seen, to a lesser degree, in control rats. Electron microscopy confirm ed the p resence of microvesicular steatosis in hepatocytes of NiCl2-treated rats (figure 4).

S e r u m E n z y m e a n d B il ir u b in A ssays

A dose-effect study of serum enzyme ac tiv itie s was p e rfo rm e d in six te s t groups (5 to 18 ra ts p e r group) th a t received sc injections of NiCl2 at dosages of 125, 250, 375, 500, 625, or 750 |xmol per kg and in a control group (14 rats) that received sc injection of the NaCl vehicle solution; the rats w ere all killed 24 hr post-injection. As shown in figure 5, significant increases of serum aspar­tate aminotransferase (AST) and alanine am inotransferase (ALT) activities w ere observed in all NiCl2-treated rats. The increases w ere dose-related; the maxi­mal (7-fold) increase of serum AST activ­ity and the maximal (3-fold) increase of serum ALT activity occurred at the 625 (xmol per kg dosage of NiCl2.

In a time-course study, six test groups (6 to 15 rats per group) w ere killed 3, 6, 18, 24, 48, or 72 hr after sc injection of N iC l2 (500 jxmol p e r kg); six con tro l groups (6 to 7 rats per group) w ere killed at corresponding intervals after sc injec­tion of NaCl vehicle solution. Since ana­lytical results in the control groups did not differ significantly, the results in the 41 control rats w ere com bined. Signifi­cant increases of serum AST and ALT activities were observed in NiCl2-treated rats at all of the intervals post-injection (figure 6). The tim e-courses of increased serum AST and ALT activities w ere par­allel, w ith maximal (7-fold) increase of serum AST activity and maximal (3-fold) increase of serum ALT activity at 24 hr after injection of NiCl2.

Dose-effect and tim e-course relation­ships for serum alkaline phospha tase activity in th e same groups of N iCl2-

112 DONSKOY, DONSKOY, FOROUHAR, GILLIES,

F ig u r e 3. Photomicrograph of liver from a rat killed 48 hr after sc injection of NiCl2 (500 jxmol per kg) to illustrate microvesicular fatty metamorphosis (arrows) (light microscopy, hematoxylin-eosin stain, x 100).

F ig u r e 4 . Electron micrograph of hepatocytes from a rat killed 4 8 hr after sc injection of NiCL ( 5 0 0 |j.mol per kg) to confirm the presence of microvesicular steatosis (arrows) (iiranvl acetate-lead citrate stain, x 1 ,7 0 0 ) .

treated rats are shown in figure 7. Signif­icant dim inution of serum alkaline phos­phatase activity was noted at 18 to 72 hr after injection of NiCl2 (500 |j.mol per kg) (figure 7, u p p e r panel). D ose-rela ted dim inution of serum alkaline phospha­tase activity was observed in rats killed 24 hr after injection of NiCl2 at dosages from 375 to 750 |xmol p er kg (figure 7, lower panel). At the h ighest dosage of NiCl2 (750 (jimol per kg), there was 72 p e rc e n t red u c tio n of serum alkaline phosphatase activity.

Nickel chloride was added in concen­trations of 10, 100, 250, and 500 ¡¿mol per L to serum specim ens from 6 control

rats. T hese in vitro additions of N iCl, did not cause significant alterations of serum alkaline p h o sp h a tase , AST, or ALT activities, indicating that abnorm al­ities of serum enzym e activities in NiCl2- treated rats are not m ediated by direct inhibition or activation of the enzymes by serum N i[II].

B iliru b in c o n c e n tra tio n s in se ru m specimens from control rats in the dose- effect and tim e-course studies averaged 0 .5 ± 0 .4 mg p e r L. No significant increases of serum bilirubin concentra­tions w ere n o te d in ra ts killed a fter injections of NiCl2 at dosages from 125 to 750 (xmol p er kg; the m ean values for

MARZOUK, REID, ZAHARIA, AND SUNDERMAN

HEPATIC TOXICITY OF NICKEL CHLORIDE 1 1 3

r̂ l Mean ± SO

* P < 0.05 *

N1CI2 dose (fi mol / kg, 24 hr)

F igure 5. Dose-effect study of aspartate amino­transferase (AST) and alanine aminotransferase (ALT) activities in serums from rats at 24 hr after sc injec­tion of NaCl vehicle (controls, N — 14) or NiCl2 (125 to 750 jjimol per kg, 5 to 18 rats per group).

serum bilirubin concentrations ranged from 0.6 ± 0.8 to 1.1 ± 0.9 mg per L.

E f f e c t s o f D ie t h y l d it h io c a r b a m a t e

S o d iu m d ie th y ld i th io c a r b a m a te (DDC), was adm inistered to rats by im injection, alone, or with simultaneous sc injection of NiCl2, at dosage combina-

r1! Mean ± SD

Controls 3 6 18 24 48 72

Hours after NiC ̂(500 n mol I kg)

F igure 6. Time-course study of AST and ALT activities in serums from control rats (N = 41) and NiCl2-treated rats (500 (xmol per kg, sc, 6 to 15 rats/ group).

^ Mean±SD

* P < 0.05

NiCI2 dose (n mol / kg, 24 hr)

F igure 7. Alkaline phosphatase activity in serums from the same groups of control and NiCl2-treated rats that are described in the captions for figures 5 and 6.

tions that have been shown to cause 6- fold increase of hepatic nickel concentra­tion and 6-fold increase of hepatic hem e oxygenase activity, com pared to rats that received NiCl2, alone.218’23 As shown in tab le I, se ru m n ickel co n cen tra tio n s were increased 3-fold in rats killed 24 hr after injection of D D C, alone, com pared to vehicle controls; serum nickel concen­trations w ere increased 1.4-fold in rats killed 24 hr after simultaneous injections of DD C and NiCl2, com pared to rats that received only NiCl2.

At 24 h r after administration of D D C, alone, serum AST activity was increased 2.5-fo ld and serum ALT ac tiv ity was increased 1.3 fold, com pared to vehicle- treated controls. Administration of D DC plus NiCl2 caused statistically insignifi­cant increases of serum AST and ALT activities, com pared to rats that received only N iC l2 (figure 8, u p p e r panels). Administration of D DC and NiCl2, sin­gly, at th e specified dosages d id not affect serum alkaline phosphatase activ-

114 DONSKOY, DONSKOY, FOROUHAR, GILLIES, MARZOUK, REID, ZAHARIA, AND SUNDERMANTABLE I

Effect of Diethyldithiocarbamate on Serum Ni Concentration in NiC^-Treated Rats*

M C I 2 ( \ im o l/k g )

DDC(m m o l/kg )

N o. o f R a ts

Serum Ni , m ean ± SD

( v g /L )

0 0 8 2.3 ± 0.6250 0 9 3,900 ± 1.900f0 1.3 8 7.0 ± 2.9t

250 1.3 10 5,400 ± 1,500$

*Nickel chloride (NiCl2 ) injected sc;Diethyldithiocarbamate (DDC) injected im;24 hours before death.

fP < 0.05 vs_ vehicle-treated controls, jp < 0.05 vs NiCl2 alone.

ity, b u t co m b in ed a d m in is tra tio n of DDC and NiCl2 acted synergistically to produce 36 percent reduction of serum alkaline phosphatase activity (figure 8, lower left panel). Com bined adm inistra­tion of D D C and NiCl2 acted synergisti­cally to produce 5-fold increase of serum bilirubin concentration (figure 8, lower right panel).

Administration of D D C , alone, caused 50 percent increase of hepatic concentra­tion of TBA-chrom ogens, com pared to vehicle-treated controls; administration of D D C plus NiCl2 did not significantly affect h ep a tic co n cen tra tio n of TBA- ch ro m o g en s, co m p a re d to ra ts th a t received only NiCl2 (table II).

DiscussionThe present findings that conjugated

dienes are increased 13-fold in hepatic microsomal lipids and that TBA-chromo­gens are increased 4-fold in hepatic cyto­sol of NiCl2-treated rats implicate lipid perox idation as a p ossib le m olecu lar mechanism for hepatocellular injury in acute Ni[II]-poisoning. The time-course and dose-effect relationships for NiCl2- in d u ced in creases of serum AST and A LT a c tiv it ie s , as o b se rv e d in th e p resent study, correspond closely to the p rev iously re p o r te d tim e-co u rse and

F igure 8. E ffects of d ie th y ld ith io ca rb am ate (DDC) and NiCl2, singly or com bined, on serum en zym e a c tiv itie s and serum bilirubin concen­tration in rats. The dosage of DDC was 1.3 mmol per kg, im, 24 hr before sacri­fice; the dosage of NiCl2 was 250 |xmol per kg, sc, 24 hr before sacrifice (10 to 18 rats per group). The asteriks indicate statistical significance (P < 0.05) in comparison with the con­trol rats (solid bars).

800-1

400-

Mean ± SD * P < 0.05

I

400-,

200 -

$ 0

80 -

e 4 0 -

1

It

| Controls 0 3 DDC (1.33 mmol I kg) S NIC|2 (250 n mol / kg) Q DDC + NiCI2

HEPATIC TOXICITY OF NICKEL CHLORIDE 1 1 5TABLE II

Effect of Diethyldithiocarbamate on Hepatic TBA-Chromogen Concentration in NiCl^-Treated Rats*

N iC l2(\xm o l/kg )

DDC (mmol / k g )

N o. o f R a ts

TBA-Chrom ogens mean ± SD

(nm ol MDA/g d r y w t)

0 0 11 0.86 ± 0.30500 0 7 2.14 ± 0.82f0 1.0 6 1.32 ± 0.75f

500 1.0 6 2.05 ± 0.74$

♦Nickel chloride (NiC^) injected sc;Diethyldithiocarbamate (DDC) injected im;18 hours before death,

fp < 0.05 vs vehicle-treated controls.^Not significantly different from NiCl2 , alone.

d o se -e ffe c t re la t io n s h ip s for N iC l2- induced increases of TBA-chromogens in liver hom ogenates.21 Hepatic lipid p er­ox idation in N iC l2- tre a te d ra ts may reflect the transient depletion of hepatic g lu ta th ione th a t occurs four to six hr after injection of NiCl2.11,23 In consider­ing the pathophysiological significance of lipid peroxidation, Younes and Siegers,30 Halliwell and G utteridge,910 and Clavel et al6 delineated three general cases: (1) lip id p erox ida tion th a t re p re se n ts an end-result of cell necrosis and autolysis,(2) lipid peroxidation that induces cell in ju ry by a c a sc a d e o f s u b s e q u e n t even ts, in c lu d in g lysosom al enzym e release and in tracellu lar Ca2 + uptake, and (3) lipid peroxidation that is inhib­ited or reversed by protective cellular systems, such as glutathione peroxidase, and by e n d o g e n o u s an d ex ogenous antioxidants. The absence of histologic evidence of hepatic necrosis in NiCl2- treated rats m ilitates against accumula­tion of TBA-chromogens and lipid conju­g a t e d d i e n e s as e n d - r e s u l t s o f hepatocellular demise. To elucidate the role of lipid peroxidation in the hepatic toxicity of NiCl2, studies are underway to d e te rm in e w h e th e r or no t N i[II]- induced lipid peroxidation and hepato­c e llu la r d am ag e a re s u p p re s s e d by administration of antioxidants.

In the present study, the morphologi­cal changes in livers of NiCl2-treated rats (500 (xmol p e r kg, sc) w ere less p ro ­nounced than those observed by M athur et al1213 following daily administration of N iS 04 to rats for one to three months. M athur et al12 noted foamy cytoplasm of hepatocytes and focal hepatic necrosis in rats tha t received 60 or 90 daily in jec­tions of N iS 0 4 (50 |xmol per kg, ip); peri­portal hypercellularity, bile duct prolif­eration , and K upffer cell hyperp lasia w ere evident at 90 days. M athur e t al13 also d e te c te d foam y cy to p la sm and vacuolization of hepatocytes, congestion and dilatation of hepatic sinusoids, and focal necrosis of hepatic parenchym a in rats that received derm al applications of N iS 04 (1.0 and 1.7 mmol per kg, daily, for one m onth). On th e o th e r hand , Yawets e t al29 did not report m orphologi­cal changes in livers of rats that received six injections of NiCl2 (170 |JLmol per kg, ip , th r ic e w e e k ly for tw o w e e k s), a lth o u g h th e N iC l2 tre a tm e n ts su p ­p re s s e d p h e n o b a rb ita l in d u c tio n of hepatic cytochrom e P-450.

The dose-related increases of serum AST and ALT activities that occurred in NiCl2-trea ted rats have not previously been reported. H epatocellular release is p ro b a b ly re s p o n s b ile for in c re a s e d serum activities of these enzym es, bu t toxic effects of NiCl2 in extrahepatic tis­sues may c o n tr ib u te to th e o b serv ed abnormalities. An unexpected outcome of the p resen t study was the finding that serum alkaline phosphatase activity was d im inished in rats that received large doses of NiCl2. Review of the literature disclosed that such hypophosphatasemia was previously noted by W hanger28 who found 45 p e rc e n t reduction of plasm a alkaline phosphatase activity in rats fed a diet containing nickel acetate (1000 ppm) for six weeks, and by W eischer e t al26 who observed 21 p ercen t reduction of serum alkaline phosphatase activity in rats exposed continuously to inhalation

11 6 DONSKOY, DONSKOY, FOROUHAR, GILLIES, MARZOUK, REID, ZAHARIA, AND SUNDERMAN

of nickel oxide aerosol (0.8 mg per m3) for 28 days. T he m echanism w hereby Ni[II] induces hypophosphatasem ia is unknown.

The influence of diethyldith iocarba­mate (DDC) on hepatic lipid peroxida­tion, serum nickel and bilirubin concen­trations, and serum enzym e activities was investigated in view of the synergis­tic effects of D D C and NiCl2 on hem e oxygenase activity and m etallothionein concentration in rat liver20,22 and the enhanced accumulation of nickel in liver and o th e r tissues of rats trea ted w ith D D C p lu s N iC l2.2,23 In th e p re s e n t study, adm inistration of D D C increased serum nickel concentrations in control and NiCl2-treated rats, bu t did not influ­ence the effects of NiCl2 on serum AST and ALT activities or hepatic concentra­tions of TBA -chrom ogens. C om bined injections of D D C and NiCl2 acted syn- ergistically to dim inish serum alkaline p hosphatase ac tiv ity and to increase serum bilirubin concentration. The lat­ter finding probably reflects synergistic induction of hem e oxygenase activity, since hem e oxygenase is the rate-limit- ing enzyme in the pathway of hem e deg­radation to b ilirubin.24

AcknowledgmentsThe authors are grateful to Sidney M. Hopfer,

P h.D ., for assistance in collecting blood from the retro-orbital venous plexus; to Eva Horak, Ph.D ., for assistance in optimizing instrumental parameters for serum enzyme assays; and to Deloritiae W eatherby for measurements of serum nickel concentrations.

References1 . B a s e l t , R. C., S u n d e r m a n , F. W., J r . , M it c iie l l ,

]., and H o r a k , E.: Comparisons of antidotal effi­cacies of sodium diethyldithiocarbam ate, D- penicillamine, and triethylenetetram ine upon acute toxicity of nickel carbonyl in rats. Res. Comm. Chem. Pathol. Pharmacol. 18:677-688,1977.

2. B e l l iv e a u , J . F ., O ’L e a r y , G. P., C a d w e l l , L . , and S u n d e r m a n , F. W ., J r .: Effect of diethyldi­

thiocarbamate on nickel concentrations in tis­sues of NiCl2-treated rats. Ann. Clin. Lab. Sci. IS:349—359, 1985.

3. B e r g m e y e r , H. V., S c h e ir e , P., and W a h l e f e l d , A. W .: Optim ization of methods for aspartate aminotransferase and alanine aminotransferase. Clin. Chem. 24:58-73, 1978.

4. B r a g d o n , J. H ., S u n d e r m a n , F. W . , J r . , and S u n ­d e r m a n , F. W . : M ethod for determ ination of total serum lipids. Lipids and the Steroid Hor­mones in Clinical Medicine. Sunderman, F. W. and Sunderman, F. W . , Jr., eds. Philadelphia, J. B . Lippincott Co., 1960, pp. 11—14.

5. B r o w n , S . S . and S u n d e r m a n , F. W ., J r . , eds: Progress in Nickel Toxicology. Oxford, Black­wells, Ltd, 1985, pp. 1-244.

6. C l a v e l , J. P., E m e r it , J., and T i iu il l ie r , A.: Lipi- doperoxidation and free radicals. Role in cellular biology and in pathology. Path. Biol. 33:61-69, 1985.

7. F a w a d e , M. M. and P a w a r , S. S.: E ffect of NiCl2, CoCl2, and cycloheximide on microsomal drug metabolism and ALA-synthetase during thiodem eton therapy. Indian J. Exper. Biol. 21:343-346, 1983.

8. F r a n k , L., W o o d , D. L., and R o b e r t s , R . J.: Effect of diethyldithiocarbamate on oxygen tox­icity and lung enzyme activity in immature and adult rats. Biochem. Pharmacol. 27:251-254,1978.

9. H a l l iw e l l , B. and G u t t e r id g e , J. M. C.: Oxygen toxicity, oxygen radicals, transition metals, and disease. Biochem. J. 219:1-14, 1984.

10. H a l l iw e l l , B ., and G u t t e r id c e , J. M. C.: Lipid peroxidation, oxygen radicals, cell damage, and antioxidant therapy. Lancet 1:1396-1397, 1985.

11. M a in e s , M . D. and K a p p a s , A.: Nickel-mediated alterations in the activity of hepatic and renal en zy m es o f h e m e m e tab o lism an d h em e dependent cellular activities. Clinical Chemistry and Chemical Toxicology o f Metals. Brown,S. S., Amsterdam, Elsevier/North Holland Bio­medical Press, 1977, pp. 75-81.

12. M a t h u r , A. K . , C h a n d r a , S. V., B e h a r i , J., and T a n d o x , S. K . : Biochemical and morphological changes in some organs of rats in nickel intoxica­tion. Arch. Toxicol. 37:159-164, 1977.

13. M a t h u r , A. K . , D a t ta , K . K . , T a n d o x , S. K . , and D ik s h it h , T . S. S.: Effect of nickel sulfate on male rats. Bull. Environ. Contam . Toxicol. 17:241—248, 1977.

14. P er r y , B . W ., D o u m a s , B . T., and B a y se , D . D . : A candidate reference method for determination of bilirubin in serum. Test of transferability. Clin. Chem. 29:297-301, 1983.

15. R e c k n a g e l , R . O. and G l e n d e , E. A., J r . : Spec- trophotom etric detection of lipid conjugated dienes. Meth. Enzymol. i05:331—337, 1984.

16. R o s n e r , B.: Fundamentals o f Biostatistics, Bos­ton, Duxbury Press, 1982, pp. 1-547.

17. S u n d e r m a n , F . W., J r . , ed.-in-chief: Nickel in the Human E nvironm ent (IARC Publ. No. 53), Oxford, Oxford Univ. Press, 1984, pp. 1-530.

18. S u n d e r m a n , F. W ., J r . , B ib e a u , L. M . , and R e i d ,

HEPATIC TOXICITY OF NICKEL CH LORIDE 1 1 7

M. C.: Synergistic induction o f microsom al hem e oxygenase activity in rat liver and kidney by diethyldithiocarbamate and nickel chloride. Toxicol. Appl. Pharmacol. 71:436-444, 1983.

19. S u n d e r m a n , F. W ., J r . , C r is o s t o m o , C., R e i d , M. C., H o p f e r , S. M., and N o m o t o , S.: Rapid analysis of nickel in serum and whole blood by electrotherm al atomic absorption spectropho­tometry. Ann. Clin. Lab. Sci. 14:232-241, 1984.

20. S underm an , F . W ., Jr. and F r a s e r , C. B . : Effects of NiCl2 and diethyldithiocarbamate on m etallothionein in rat liver and kidney. Ann. Clin. Lab. Sci. 13:489-495, 1983.

21. S u n d e r m a n , F. W ., J r . , M a r z o u k , A., H o p f e r ,S . M., Z a h a r ia , O., and R e i d , M. C.: Increased lipid peroxidation in tissues of nickel chloride- treated rats. Ann. Clin. Lab. Sci. i5:229—236, 1985.

22. S u n d e r m a n , F. W ., Jr., R e i d , M. C ., B ib e a u , L . M ., and L i n d e n , J. V.: Nickel induction of microsomal heme oxygenase activity in rodents. Toxicol. Appl. Pharmacol., 68:87-95, 1983.

23. S u n d e r m a n , F. W ., J r . , Z a h a r ia , O . , R e i d , M. C., B e l l iv e a u , J. F., O ’L e a ry , G . P., Jr., and G r if f in , H . : Effects of diethyldithiocarbamate and nickel chloride on glutathione and trace metal concentrations in rat liver. Toxicology 32:11-21, 1984.

24. T e n h u n e n , R . , M a r v e r , H . S . , and S c h m id , R . :

Microsomal hem e oxygenase. Characterization of the enzyme. J. Biol. Chem. 244:6388—6394,1969.

25. T i e t z e , N. W ., R in k e b , A. D., and S h a w , L. M.: IFCC method for alkaline phosphatase. J. Clin. Chem. Clin. Biochem. 21:731—748, 1983.

26. W e is c h e r , C. H . , K o r d e l , W ., and H o c h r a i .n e r ,D.: Effects of NiCl2 and NiO in Wistar rats after oral uptake and inhalation exposure respec­tively. Zbl. Bakt. H y g . I. Abt. Orig. B 171:336- 351, 1980.

27. W e s t B. and S u n d e r m a n , F. W . : Nickel poison­ing. VII. The therapeutic effectiveness of alkyl d ith io carbam ates in ex perim en ta l anim als exposed to nickel carbonyl. Amer. J. Med. Sci. 236:15-25, 1958.

28. W h a n g e r , P. D .: Effects of dietary nickel on enzyme activities and mineral contents in rats. Toxicol. Appl. Pharmacol. 25:323—331, 1973.

29. Y a w e t s , A . , A l t e r , A . , and O r o n , U . : Biochemi­cal and histological anomalies in rat hepatic tis­sue following administration of bichromate and nickel in ionized form. Toxicology 33:145—155,1984.

30. Y o u n e s , M. and S i e g e r s , C-P.: In terre la tio n betw een lipid peroxidation and other hepato- toxic events. Biochem. Pharmacol. 33:2001- 2003, 1984.