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A New Evaluation of Peroxisome Proliferation in Rainbow Trout Hepatocytes in Culturea CHRISTIAN FIGUEROA, MARfA E. KAWADA, CECILIA KOENIG,

AND MANUEL J. SANTOSb

Departamento de Biologia Celular y Molecular Facultad de Ciencias Biolbgicas Pontificia Univevsidad Catdlica Casilla 114-0, Santiago, Chile

Peroxisomal proliferation is a species-specific phenomenon. Recently this phenome- non has been evaluated in teleosts. Rainbow trout exposed to hypolipidemic drugs in vivo show a significant increase in their liver P-oxidation system. l s 2 Furthermore, isolated rainbow trout hepatocytes in primary culture incubated in vitro with these drugs also showed an increase in peroxisomal P-oxidation, although this increase was at a very low statistical ~ignificance.~ However, peroxisome proliferation in cul- tured trout hepatocytes has not been examined at the morphological level by electron microscopy. In order to accomplish this, we incubated isolated rainbow trout hepato-

TABLE 1. Effect of Peroxisome Proliferators on Peroxisomal and Mitochondria1 Enzymes in Primary Cultures of Rainbow Trout Hepatocytes

Enzyme Activities

Mitochondria Peroxisomes

Catalase FA0 cytox Treatment (Uimg protein) (mUimg protein) (mUimg protein)

Control 0.496 * 0.026 (14)

2 mM Clo 0.603 * 0.045 (10)

2.75 mM Clo 0.592 f 0.008 ( 5 )

4 mM Clo 0.577 * 0.055 (6)

1 mM 2,4-D 0.568 i 0.029 (6) (115%)

( 1 OO?Lo)

(122%)

(119%)

(1 16%)

5.299 A 0.494 (15)

5.074 * 0.650 (1 1) (96%)

5.231 i 0.885 ( 5 ) (99Yo)

6.206 i 0.918 (6)

4.297 f 1.008 ( 5 )

(100%)

( 1 17%)

(81%)

12.911 k 0.575 (14)

13.072 * 0.940 (8)

12.412 .i: 1.398 (4) (96%)

11.647 f 0.879 (5) (90%)

15.157 i 1.092 (6) (117%)

(100%)

(10 1 Yo)

Clo, clofibrate; 2,4-D, 2,4-dichlorophenoxy acetic acid; FAO, fatty acid oxidase; Cytox, cy- tochrome c oxidase.

NOTE: Values are expressed as mean i SE; they are nonsignificant a t p < 0.05. Parentheses at right of values indicate the number of experiments; parentheses below indicate percentage with respect to control.

"This work was supported by FONDECYT (Chile), grant 1940686. bE-mail: msantos@genes.bio.puc.cl

122

FIGURE 1. Electron microscopy catalase cytochemistry of trout hepatocytes in culture incu- bated with peroxisome proliferators. (A) Control hepatocytes and (B) hepatocytes incubated with 1 mM 2,4-D for 48 h. Catalase cytochemical staining was carried out using a very short in- cubation in the diaminobenizidine medium to differentiate peroxisomes from other electron- dense vesicles. Control experiments in the absence of H2O2 or in the presence of aminotriazole were performed. P, peroxisomes; m, mitochondria. (Original magnification: 10,000~; reduced to 859'0.)

724 ANNALS NEW YORK ACADEMY OF SCIENCES

cytes in culture with known peroxisomal proliferators and characterized them bio- chemically and morphologically.

We isolated hepatocytes from male rainbow trout (Onchorynchus mykiss) and cul- tured them in the presence of the hypolipidemic drug ( 0 4 mM clofibrate) or the pes- ticizer (0-1 mM 2,4-dichlorophenoxy acetic acid; 2,4-D). After 48 hours of incuba- tion, marker enzymes were determined in the hepatocyte homogenates. As seen in TABLE 1, no significant increase was found in peroxisomal (catalase and fatty acid CoA oxidase) or mitochrondrial enzymes (cytochrome c oxidase). Peroxisomal pro- liferation-associated polypeptides of 80 kDa4 and 64 kDa5 were not detected in SDS- PAGE protein profiles from the treated hepatocyte homogenates.

Peroxisomes were readily detected in the cultured hepatocytes by a modified method to demonstrate catalase cytochemistry6 (FIG. 1). In control hepatocytes we found aperoxisomal volume of 1.71 x lo3 f 0.32 x lo3 cm3 and a density of 0.195 f 0.034 peroxisomes/cm2. Upon treatment with 1 mM 2,4-D the peroxisomal volume (1.52 x lo3 f 0.39 x lo3 cm3) and density (0.193 f 0.052 peroxisomes/cm2) showed no significant change. Similar results were also obtained upon treatment with clofi- brate.

In conclusion, rainbow trout hepatocytes in culture, incubated with clofibrate or 2,4-D, showed no evidence of peroxisomal proliferation or induction of peroxisomal proteins.

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