Acute effects of 2-nitropropane on rat liver and brain

Toxicology Letters, 9 (1981) 237-246 Elsevier/North-Holland Biomedical Press

ACUTE EFFECTS OF 2-NITROPROPANE

237

ON RAT LIVER AND BRAIN

ANTTI ZITTING, HEIKKI SAVOLAINEN and JUHA NICKELS* Department of Industrial Hygiene and Toxicology, and *Department of Occupational Medicine, Institute of Occupational Health, Haartmaninkatu I, SF-00290 Helsinki 29 (Finland) (Received April 2nd, 1981) (Accepted April 18th, 1981)

SUMMARY Intraperitoneal injection (50 mg/kg) of 2-nitropropane (2-NP) induced lipid accumulation, centrilobular necrosis, degranulation of rough endoplasmic reticulum, proliferation of smooth endoplasmic reticulum and mitochondrial abnormalities in rat liver 24 h after exposure. These pathological changes were accompanied by elevated serum alanine aminotransferase (ALAT) levels. Hepatic glutathione content increased rapidly in exposed rats. 2-NP depressed markedly hepatic cytochrome P-450 and microsomal monooxygenase activity while the enzymes, epoxide hydratase, UDPglucuronosyltransferase and cytosolic glutathione peroxidase were enhanced. 2-NP caused an increase of acetylcholine esterase activity in the brain. This effect was also detected in synaptosomes isolated from exposed rats. The results suggest peroxidative damage in the cells.

INTRODUCTION

Solvent systems containing 2-NP possess several desirable properties e.g. reduced drying time, mild odor, more complete solvent release and greater wetting ability. 2NP is used in coatings, printing-inks, adhesives and as a solvent in food processing, thus occupational exposure may occur in several industries [ 11. Nitroparaffins cause liver damage regardless of the route of administration [2]: recently it has been shown that 2-NP is carcinogenic in the rat when administered by inhalation [3]. Although little information is available on the metabolism of nitroparaffins, the liver has been implicated as the principal site of 2-NP metabolism [4]. 2-NP is degraded to acetone and nitrite by the microsomal cytochrome P-450 system [5]. 2-NP reacts nonenzymatically with reduced glutathione and in addition is a substrate for the glutathione S-transferases [6]. We injected rats Abbreviations: ALAT, alanine aminotransferase; GSH, liver glutathione; 2-NP, 2-nitropropane; rough endoplasmic reticulum; SER, smooth endoplasmic reticulum. 0378-4274/81/0000-0000/$02.50

0 Elsevier/North-Holland

Biomedical Press

RER,

238

i.p. with 2-NP and studied its acute effects on some hepatic drug-metabolizing enzymes and compared them with effects on some biochemical parameters in the brain. MATERIALS AND METHODS

Male Wistar rats (300-320 g) were injected i.p. with 2-NP (50 mg/kg) in olive oil. After killing the animals by decapitation, samples of liver, kidneys, brain, and blood were obtained. Liver glutathione (GSH) was determined by the method of Saville [7], Ca2+-aggregated microsomes were prepared [B] and analyzed for cytochrome P-450 [9], cytochrome bs [9], 7-ethoxycoumarin 0-deethylase [lo], 7ethoxyresorufin 0-deethylase [ 111, NADPH-cytochrome c reductase [ 121, UDPglucuronosyltransferase (4.5 mM UDP-glucuronic acid, 0.35 mM p-nitrophenol) [ 131, and epoxide hydratase [14]. Glutathione peroxidase [15] and glutathione Stransferase (1 mM 1-chloro-2,4_dinitrobenzene) [6] were measured in the liver cytosol. Acid proteinase activity in the cerebral homogenate was assayed by the method of Marks et al. [16]. The activities of acetylcholine esterase [17] and 2’ ,3’ -cyclic nucleotide 3’-phosphohydrolase [18] were also analyzed and the RNA content determined [19]. Protein was assayed by the method of Lowry et al. [20]. Synaptosomes were isolated by gradient ultracentrifugation [17], and the acetylcholine esterase activity determined. An aliquot was taken for electronmicroscopic examination (Fig. 1). Biopsies of the medial lobe of the liver were taken from 6 exposed rats and two controls. The samples were embedded in Epon for electron microscopy. After glutaraldehyde and osmium tetroxide fixation sections 0.5 pm thick were stained with toluidine blue and examined light-microscopically.

TABLE I EFFECT OF 2-NITROPROPANE

ON HEPATIC GLUTATHIONE Control (N = 4)

GSH nmoI/g tissue

GSH nmol/g tissue

5.79 + 0.13

1 h after exposure (N = 4) 6.55 f 0.32a

Control (N = 5)

4 h after exposure (N = 5)

24 h after exposure (N = 5)

6.27 + 0.16

9.13 f 0.53b

11.87 + 2.02b

Values are the means + SD. Statistical evaluations were performed using Student’s t-test. aP < 0.01. “P < 0.001.

The fraction

of myelin and cellular

ultracentrifugation.

Note the absence

by gradient

vesicles.

isolated

and synaptic

Fig. 1. Synaptosomes

mitochondria

some free mitochondria,

fragments.

contains

and typical

synaptosomes

which contain

e.g.

3.96 zk0.55 6.34 rf 1.Ol“ 6.85 zk 1.26~

nmol/min/mg

prot.

Epoxide hydratase

UDP-glucuronosyltransferase nmol/min/mg prot.

1.83 k 0.41 3.71 + 0.72c 4.52 + 0.73C

0.43 + 0.06 0.50 + 0.03 0.45 rt:0.04

0.66 * 0.05 0.51 2 0.09b 0.37 * 0.03c

0.82+0.11 0.86 f 0.07 0.69 + 0.06a

Glutathione S-transferase pmol/min/mg

93 * 18 102 2 IO 123 & l@

prot.

NADPH-cytochrome c reductase nmol/min/mg prot.

METABOLISM

Values are the mean of 5 animals + SD. Statistical evaluations were performed using Student’s l-test. aP < 0.05. bP < 0.01. CP < 0.001.

Control 4 h after exposure 24 h after exposure

Control 4 h after exuosure 24 h after exposure

Cytochrome b5 nmol/mg prot.

ON RAT LIVER XENOBIOTK

Cytochrome P-450 nmol/mg prot.

EFFECTS OF 2-NITROPROPANE

TABLE II

33.5 rt 3.6 41.9 + 4.Ob 49.9 f 5.9

Glutathione peroxidase nmol/min/mg

1.15+0.12 0.91 f 0. lob 0.65 + 0.03C

prot.

‘I-Ethoxycoumarin 0-deethylase nmol/min/mg prot.

81 I21 61 r1;29 33 f 12b

7-Ethoxyresorufin 0-deethyiase ~mol/min/mg prot.

241 RESULTS

The i.p. injection of 2-NP raised the amount of GSH in the liver 1 h after treatment, and the GSH content almost doubled during the following (Table I). Hepatic cytochrome P-450 levels were strongly depressed: 24 h after the treatment, they were only 56% of the control values. The amount of cytochrome bs was not affected (Table II). Concomitantly with the decrease of cytochrome P-450, the 7ethoxycoumarin and 7-ethoxyresorufin O-deethylase activities were diminished (Table II). The treatment only slightly influenced NADPH-cytochrome c reductase activity, but the microsomal epoxide hydratase and UDP-glucuronosyltransferase activities were greatly enhanced (Table II). 2-NP injection somewhat reduced the cytosolic glutathione S-transferase activity but induced glutathione peroxidase (Table II). Accumulation of lipid material was seen in the hepatocytes and in the Kupffer cells, 4 h after 2-NP treatment, and the accumulation continued for the next 20 h. Lipid droplets were the most prominent in the periportal areas (Fig. 2). In electron micrographs 4 h after 2-NP exposure, the RER showed degranulation while there was proliferation of the SER. After 24 h, necrosis of the hepatocytes around the central vein became evident (Fig. 3). The RER had almost disappeared, and the SER was vacuolated in some hepatocytes and compact in others (Fig. 4). Some cells had normal mitochondria but swollen mitochondria without cristae were also seen (Fig. 4). Necrosis was reflected in serum ALAT activities which during 24 h increased from the control value of 60 f 8 U/l ( f SD, N = 5) to 233 _+220 (N = 5) (2 p = 0.008, Cramer-von Mises two-sample two-sided test was used because of large SD). The most pronounced neurochemical effect was a significant increase of cerebral acetylcholine esterase activity (Table III). This effect was also observed in isolated

TABLE EFFECTS

III OF 2-NITROPROPANE

ON RAT BRAIN

RNA gg/mg

prot.

Acetylcholine

2’ ,3’ -cyclic

esterase nmol/min/mg

nucleotide

prot.

hydrolase

Acid proteinase nmol/min/mg

3’-phosphomin/mg

prot. pmol/

prot.

Control 4 h after exposure

13.6 f 0.8

120 + 18

9.7 + 0.6

87 + 7

12.9 k 0.8

141 + 12a

9.4 * 0.7

80 + 4a

24 h after exnosure

13.2 + 0.8

192 + 24b

9.0 + 0.Y

84 k 9

The figures are the means Student’s f-test.

aP < 0.05. bP< 0.001.

of five animals

+ SD. The statistical

evaluations

were performed

using

24 h after

vein. Hepatocytes

show small,

2-nitropropane

micrograph

Fig. 3. Electron

visible near a central

toluidine

0.5 lrn section,

x 540.

24 h after 2nitropropane

blue.

Fig. 2. Light micrograph

dark-stained

lipid droplets.

x 1360.

cell material

heavy fat infiltration

Necrotic

shows

treatment.

treatment

(arrows)

a normal

portal

by inflammatory

around

surrounded

of hepatocytes

are

Epon embedding,

cells and erythrocytes

area.

Fig. 4. Electron micrograph 24 h after 2-nitropropane treatment. The hepatocyte on the left shows tightly packed smooth endoplasmic reticulum (SER) and normal mitochondria. The hepatocyte on the right shows vacuolated endoplasmic reticulum and swollen mitochondria. x 10000.

244

synaptosomes nmol/min/mg test).

in which, 18 h after injection, the esterase activity (628 + 19 prot.) exceeded the control value of 590 + 12 (P < 0.01, Student’s t-

DISCUSSION

The rapid increase of hepatic non-protein sulfhydryl group content after 2-NP treatment can be explained as a rebound effect, which has been observed after administration of GSH depleting agents, e.g. 2-chloroethanol [21]. In addition to participating in conjugation reactions, GSH has other important protective roles. It protects against peroxidative damage by scavenging hydrogen peroxide [22] and by inhibiting lipid peroxidation in glutathione peroxidase-dependent reactions [23]. That lipid peroxidation occurs after 2-NP treatment is obvious when the changes of hepatic enzyme activities and the histopathological findings are considered. The decrease in cytochrome P-450 and the reduction in the monooxygenase activities is in accordance with similar observations, e.g. carbon tetrachlorideinduced lipid peroxidation [24]. Carbon tetrachloride increases the microsomal UDP-glucuronosyltransferase activity [24], an effect also observed with 2-NP. The probable reason for this enhancement is to be found in changes of the lipid composition of membranes, which fact may also explain the increase of the membrane-bound epoxide hydratase activity. The intensification of the hepatic glutathione peroxidase activity can be interpreted as a reaction to peroxidation. The histopathological changes are also indicative of lipid peroxidation. Heavy lipid accumulation, centrilobular necrosis, RER degranulation, SER proliferation, and mitochondrial abnormalities are identical with the effects of carbon tetrachloride in the rat (25). The causes of lipid peroxidation in 2-NP exposure are obscure. Several possibilities, however, can be postulated. Firstly, the nitrite formed from 2-NP could produce hypoxia through interference with heme [27]. In hypoxia, electrons can be shunted from the mitochondrial respiratory chain to other systems, e.g. cytochrome P-450. This results in the univalent reduction of oxygen and creates a highly reactive species [26]. An increased production of reactive oxygen species has also been observed in mitochondria after administration of uncouplers of oxidative phosphorylation [28]. The second possibility is the formation of other reactive free radical intermediates in the cytochrome P-450-dependent metabolism of 2-NP in analogy with the creation of the CC13 radical from carbon tetrachloride [29]. In addition, the incubation of rat liver slices with nitrite produces an EPR signal which is a sign of free radical production [30]. Very little is known about the activity of nitroreductase towards nitroalkanes but these reactions must be regarded as another possible source of peroxidation. In aerobic conditions, nitroreductase first forms a nitro-anion free radical which then

245

reacts with molecular oxygen, and a superoxide anion is formed together with the parent substrate [31]: similar to the proposed mechanism of paraquat [32], The neurochemical findings suggest that, even in the brain, 2-NP exerts its deleterious effect through metabolic activation. The oxidative capacity of the general brain cell population is low [33] while considerable cytochrome P-450 concentration can be found in the cerebral mitochondria 1351. Mitochondria also carry considerable nitroreductive capacity [34]. Mitochondria are especially numerous in the nerve cells, and are in intimate contact with neuronal membrane in the synapses and which were isolated as synaptosomes (Fig. 1). Synaptic membranes also contain an abundance of’ acetylcholine esterase. The increase in the acetylcholine esterase activity may therefore indicate that mitochondrial metabolism produces highly reactive species, which attack the proximate synaptic membrane. This would explain the minor effects on the metabolism in RNA and acid proteinase and the escape of the oligodendroglia-myelin system verified the unaltered 2’,3’cyclic nucleotide 3’-phosphohydrolase activity. A general disarrangement in the synaptic membranes might explain the CNS symptoms in 2-NP toxicity. ACKNOWLEDGEMENTS

We wish to thank Ms. Helena Kivisto (MSc.), Ms. Tuula Korhonen, Peltonen, and Ms. Tuula Stjernvall for technical assistance.

Ms. Ulla

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