Microbody proliferation in liver induced by nafenopin, a new hypolipidemic drug: Comparison with CPIB

BIOCHEMICAL

Vol. 52, No. 2, 1973

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

MICROBODY PROLIFERATION IN LIVER INDUCED BY NAFENOPIN, A NEW HYPOLIPIDEMIC DRUG: COMPARISON WITH CPIB Janardan

Reddy,

Donald

Svoboda

and Daniel

Azarnoff

Department of Pathology and Oncology, and Clinical Pharmacology and Toxicology Center University of Kansas Medical Center Kansas City, Kansas 66103 Received

April

5,1973

SUMMARY. Nafenopin (2-methyl-2]P-(1,2,3,4-tetrahydro-1 naphthyl) phenoxylpropionic acid, a phenolic ether with hypolipidemic properties, when administered by gavage at 100 mg/kg b wt daily for 1 to 2 weeks, caused a significant increase in the number of microbody profiles and simultaneous increase in catalase activity in livers of male rats. The concentration of catalase protein and the rate of incorporation of H3-&aminolevulinic acid into catalase fraction, as determined by immunochamical methods were approximately twice that of controls. The microbody proliferation resulting from nafenopin treatment was comparable to that induced by CPIB. Microbodies originally

in kidney

mammalian several acid

cell

tions,

in

organelles

drug,

other

(10).

resulting hypolipidemic

proliferation (Su-13437;

Copyright 111 tights

0 I973

(11).

uricase,

liver

possess

d-amino (5).

profiles

are rarely

relatively

unaffected

a significant rats

microbody in catalase

in rat

acid

and in both

proliferation activity

sexes

by most

resulting

The relationship

between

from CPIB treatment agents

examined

We now report

from

failed

by Academic Press. in arty form

Inc. reserved.

rate

a hypolipi-

mice

is not

with

The marked

of synthesis

since

microbody drug,

of

and hypo-

understood,

to induce

profiles

(6-9).

proliferation

a new hypolipidemic

537

manipula-

of microbody

was associated

microbody

however,

to date that

in number

enhanced

appears

experimental (CPIB),

liver

and

a-hydroxy

and it

of acatalasemic

in rat

catalase

in the number

encountered

increase

other

oxidase,

Alterations

2-methyl-2-[P-(1,2,3,4-tetrahydro-l-naphthyl)-phenoxyJ-propionic

of reproduction

in several

ethyl-a-p-chlorophenoxyisobutyrate

of male

enzyme

lipidemia

with

described

recently

dehydrogenase

demonstrated

CPIB-induced elevation

including

are

organelles

and identified Microbodies

of microbody

the livers

this

enzymes

Studies

demic

(l-4).

and isocitrate

or morphology these

are cytoplasmic

and liver

types

oxidative oxidase

that

(peroxisomes)

nafenopin

BIOCHEMICAL

Vol. 52, No. 2, 1973

acid)

(12,13)

induces

files

in male

rat

and of catalase

a significant

liver.

ether

Parallel

studies

increase

A simultaneous

protein

phenolic

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

was also

and these with

observed.

two compounds

CPIB are

Gilroy,

Calif.)

Nafenopin

weighing

was administered

b. wt.,

as 1% finely

stomach

tube

to four

animals

were

Ultrastructural liver l-2

hours

in

fixation

the

ed in epoxy lead

2% osmium tetroxide tissues resin.

hydroxide of catalase

tally

at 25'C

Protein

prepared

Quantitation

to the procedure

with

of catalase

nafenopin

Incorporation acid

protein

or CPIB for

--et al

catalase in

livers

activity

Heights,

Illinois.

acid

300 me/m mole) Control

small

daily

by

b. wt.

Two

or CPIB.

segments were

of rat

fixed

g-collidine.

of alcohols

was assayed

for

After and embeddstained

with

of Lowry

rats

spectrophotometri-

extracts

of Ganschow

was purified

and anticatalase

into

foot

pads

of normal

according serum was pre-

of rabbits

rats

catalase: from treated

(10).

and of rats

by immunotitration

and rats

(15).

(16).

(17)

into

of liver

and Schimke

et al

catalase

was obtained

538

of 100 mglkg

an ultramicrotome,

14 days was performed

of H3-Gaminolevulinic

(specific

Arlington

the purified

study

on deoxycholate

Rat liver

by Price

experiments.

microscope.

activity (14)

these

CPIB was given

series

cut with

to the method

protein:

outlined

by injecting

Quantitation

were

was done by the method

of catalase

a dose

to pH 7.4 with

in an electron

according

in

or CPIB treatment

in graded

by Luck

1).

Inc.,

and 14 days on nafenopin

buffered

Catalase

as described

a

(Fig.

in a dose of 250 mg/kg

nafenopin

sections

activity:

estimation

pared

Thin

in

microscopic

dehydrated

and examined

Assay

homogenates

were

is

Laboratories

used

solution.

1,3,7,10

during

(Simonson

tube

oil

For electron

intervals

CPIB,

related

grams were

olive

after

studies:

at selected

in

killed

rats

aqueous

as a 5% solution

like

activity

comparison.

by stomach

homogenized

catalase

pro-

AND METHODS

140-175

daily

of microbody

liver

structurally

for

male F-344

between

in

Nafenopin,

are

included

Inbred

of animals:

the number

increase

MATERIALS Treatment

in

treated method

H3-b-aminolevulinic Amersham/Searle, with

either

(15).

BIOCHEMICAL

Vol. 52, No. 2, 1973

NAFENOPIN

Figure

WJ -13437)

1.

AND BIOPHYSICAL

CB

Structural

RESEARCH COMMUNICATIONS

CH3 CPIB

formulas

of Nafenopin

and CPIB.

Figure 2. Portion of a liver cell from a male rat treated with Nafenopin (100 mg/kg b wt; daily by gavage) for 10 days. Marked increase in number with considerable variation in size of microbody profiles (mb) is evident. Nucleoid or crystalline core (n). X 14,500.

nafenopin

or CPIB for

14 days were

in a dose of 75 nc/lOO extract

of liver

serum were described

gm body weight

homogenate

obtained

injected

and killed

2 hours

and immunoprecipitate

and the radioactivity

previously

intraperitoneally

with later.

isotope

Deoxycholate

of catalase

of catalase

the

with

fraction

anticatalase

determined

as

(10). RESULTS

Increase endoplasmic the initiation the microbody

in

the number

reticulum

were

of nafenopin profiles

in

of microbody evident

in the

treatment. liver

profiles

cells

liver

During were

539

and proliferation cells

within

the second

numerous

l-3

of smooth days

after

week

on nafenopin,

and revealed

considerable

BIOCHEMICAL

Vol. 52, No. 2, 1973

Table 1. Nafenopin

Liver weight and CPIB.

Groupa

and catalase

No. of rats

Untreated control Nafenopin (14 days) CPIB (14 days)

variation

in size with

by CPIB.

The data

and catalase

7.8 6.2

significant

weight

activity

of rats

treated

nafenopin

suggest

that

nafenopin

fraction

per

from

normal

rat

the rates

incorporation

gm of liver

of labeled

precursor

catalase

catalase rats

liver

540

rats

from

was 0.22

ml and 0.23

in

extracts

into

synthesized

ml.

These

of rats

and

to CPIB

catalase

the assumption

each newly

from

In order

in nafenopin acid

1 ml of

catalase

to be 0.115 liver

of catalase

with

treated

The anticatalase

that of controls.

at 14 days, into

in male

activity

was found

twice

incorporated

catalase corresponded

3.

of H3-6-aminolevulinic

was studied

produced The increase

to precipitate

protein

of synthesis

weight

activity.

2 weeks

induced

profiles.

needed

of catalase

to that

compounds

shown in Figure

of antiserum

in rats

The liver

of liver

or CPIB was approximately

the

Both

1.

of microbody

the

administered

and CPIB on liver

and CPIB treated

the amount

and compare rats,

amount

extract

comparable

or CPIB for

are

were

9.6

proliferation

nafenopin.

completely

The amount

1 ml of 5% liver

treated

2 weeks

from nafenopin

ml respectively.

determine

with

the number

to precipitate

b wt)

and in catalase

nafenopin

and CPIB for

extract

results

weight

with in

in Table

of immunoprecipitation

serum required

given

in liver

to the increase

5% liver

presented

activity protein)

85 -+

was roughly

of nafenopin

was more pronounced

The results with

are

increases

in liver

closely

on the effects

with

39 + 3.6 gl? 6.4

The microbody

two weeks

treated

T 0.38 3 0.35

and CPIB (250 mg/kg

2).

rats

weight Catalase gm body wt) (units/mg Mean -+ standard error

3.8 + 0.31

(Fig.

for

activity

Liver (gm/lOO

RESEARCH COMMUNICATIONS

in male

4

and shape

nafenopin

activity

3 3

aNafenopin (100 mg/kg b wt) daily by stomach tube.

treated

AND BIOPHYSICAL

that

the

catalase

BIOCHEMICAL

Vol. 52, No. 2, 1973

0.04

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

0.06 0.12 0.16 0.20 ml OF ANTICATALASE SERUM ADDED I ml OF LIVER EXTRACT

0.24 TO

Figure 3. Immunoprecipitation of liver catalase in male rats treated with Nafenopin and CPIB for 2 weeks. Increasing amounts of anticatalase serum were added to constant quantities of deoxycholatetreated liver extracts from normal (A-A), Nafenopin (o-o) and CPIB (a-o) treated rats. The resulting immunoprecipitates were sedimented and the catalase activity in the supernatant was determined by the spectrophotometric method. Dashed lines are extrapolations of the linear portions of the titration curves to zero catalase activity. Nafenopin (100 mg/kg b wt) and CPIB (250 nglkg b wt) were administered daily by gavage.

molecule

is

2 shows

the same in normal,

the results

increase

of this

in radioactivity

and CPIB when

compared

nafenopin

experiment of catalase

and CPIB-treated and it

is

fraction

rat

evident of rats

livers.

that

there

treated

with

Table is

a marked

nafenopin

to controls. DISCUSSION

Unlike liver

cells

other are

manipulations compounds

cytoplasnic infrequently

(1,19).

constituents, altered

microbodies

in number

or morphology

CPIB and acetylsalicylic

known

to induce

significant

profiles

in liver

cells

response

is more pronounced

(6-9,18,19). when

acid

increase

in

541

to that

are

two

of microbody

microbody

resulting

in

by experimental

the only

the number

The CPIB-induced compared

(peroxisomes)

from

proliferative the administra-

BIOCHEMICAL

Vol. 52, No. 2, 1973

Table liver

AND BIOPHYSICAL

2. Comparison of radioactivity of catalase fraction in male rats treated with nafenopin and CPIBa.

Groupb Control Nafenopin CPIB aH3-6-ami nolevulinic acid intraperitoneally 2 hours from the liver extract by radioactivity of catalase bNafenopin by gavage

tion

liferation

and the regulatory

proliferation

mechanism(s)

properties

effect

The present inducing

proliferation

of catalase in

the

these

studies

body proliferation Nafenopin

livers

in the

liver

induced

because

pro-

by CPIB events

between

suggested

CPIB treatment

daily

of these

relationship

studies

given

of microbody

initiation

additional

microbody

of the hypothat

the

micro-

may be independent

ether

which

of male

treated

rats.

in male

rats.

compound

of microbody

structurally

of its

Nearly

nafenopin

similar

two-fold

as effective

enlargement

14 days.

Nafenopin,

elevation

It

in catalase

in

were

the quantity

also is

produced

observed

evident

from

as CPIB in inducing hepatic micro-

of the

542

of

to CPIB,

increase

of catalase for

capable profiles.

and concomitant

of synthesis

with is

marked

is

profiles

nafenopin

causes

another

in the number

and the rate

of rats that

in

was invoked

of microbody

protein

livers

synthesis

identifies

increase phenolic

in the

significance

a possible

from

were

(7,ll).

significant

activity

b wt)

metabolism

investigation

a hypolipidemic rapid

Although

resulting

hypolipidemic

1 2.1 1.9

involved

of CPIB,

body proliferation

6,552 13,942 12,528

The biological

and cholesterol

lipidemic

Ratio

of catalase

to be elucidated.

gm of

dpm

and CPIB (250 mg/kg

acid.

and stimulation

per

75 pc per 100 gm of body weight was given before sacrifice. Catalase was precipitated the addition of anticatalase serum and the per gm of liver was calculated.

(100 mg/kg b wt) for 14 days.

of acetylsalicylic

remain

RESEARCH COMMUNICATIONS

liver

presumably

resulting

BIOCHEMICAL

Vol. 52, No. 2, 1973

from hypertrophy hepatomegaly body

and hyperplasia may, in part,

profiles

present

cells

endoplasmic

(20).

to the marked reticulum

The nafenopin

induced

proliferation

of micro-

in liver

cells

observed

in

the

study.

capable

of chemicals

of producing

role

necessary

experiments

microbody

of microbodies

in

to delineate

the

and hypolipidemic

portion

of liver

be attributed

and smooth

Identification

the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

are

of the

two hypolipidemic

changes

agents

proliferation

cellular

in our

configuration is

necessary

metabolic is

if

states

essential

metabolism.

relationship, resulting

in progress chemical

and altered

from nafenopin laboratory

between

are

in understanding

Additional any,

which

studies

are

microbody

proliferation

and CPIB treatment.

in an attempt

Further

to determine

which

of the respective

molecules

of these

to induce

microbody

proliferation.

hepatic

This work was supported by USPHS grants GM-15956 and CA-5680. The drugs used in this investigation were generously supplied by Dr. William E. Wagner, CIBA pharmaceuticals, Summit, N. .I. (Nafenopin) and by Dr. J. Noble, Ayerst Laboratories, New York, N. Y. (CPIB). REFERENCES 1. 2. 3. 4. 5. 6. 7. a. 9.

10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

Hruban, 2. and Rechcigl, M., Jr., Int. Rev. Cytol. (Suppl, l), 1969. Reddy, J. and Svoboda, D., J. Histochem. Cytochem. 20:140, 1972. Novikoff, P. M. and Novikoff, A. B., J. Cell Biol. 53:532, 1972. Hruban, Z., Vigil, E. L., Slesers, A. and Hopkins, E., Lab. Invest. 27: 184, 1972. Leighton, F., Poole, B., Lazarow, P. B. and DeDuve, C., J. Cell Biol. 41:521, 1969. Hess, R., Staubli, W., and Riess, W., Nature 208:856, 1965. Svoboda, D. J. and Azarnoff, D. L., J. Cell Biol. 30:442, 1966. Reddy, J., Bunyaratvej, S. and Svoboda, D., J. Cell Biol. 42:587, 1969. Reddy, J., Bunyaratvej, S. and Svoboda, D., Amer. J. Path. 56:351, 1969. Reddy, J., Chiga, M., and Svoboda, D., Biochem. Biophys. Res. Commun. 43:318, 1971. Azarnoff, D. L. and Svoboda, D. J., Arch. Int. Pharmacodyn. 181:386, 1969. Hess, R. and Bencze, W. L., Experientia 24:418, 1968. Dujovne, C. A., Weiss, P., and Bianchine, J. R., Clin. Pharmacol. Ther. 12:117, 1971. Luck, H., Catalase in Methods of Enzymatic Analysis, ed. by Bergmeyer, H., P. 885. New York, Academic Press. Inc., 1963. .J.-Biol. Chem. 244:4649, 1969. Ganschow, R. E. and Schimke, R. T;, Lowry, 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., J. Biol. Chem. 193:265, 1951. Price, V. E., Sterling, W. R., Tarantola, V. A., Hartley, R. W., Jr. and Rechcigl, M., Jr., J. Biol. Chem. 237:3468, 1962. Lab. Invest. 15:1884, 1966. Hruban, Z., Swift, H. and Slesers, A., J. Cell Biol 35~127, 1967. Svoboda, D., Grady, H., and Azarnoff, D., Toxic. Beckett, R.B., Weiss, R., Stitzel, R.E. and Cenedella, R.J., Appl. Pharm. 23:42, 1972.

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