THE JOURNAL OF HISTOCHEMISTRY AND CYTOCHEMISTRY Vol. 21, No. 11, pp.

967-971, 1973
Copyright © 1973 by The Histochemical Society, Inc. Printed in U.S.A.

POSSIBLE PROPERTIES OF MICROBODIES (PEROXISOMES)

MICROBODY PROLIFERATION AND HYPOLIPIDEMIC DRUGS”2

JANARDAN K. REDDY
Department of Pathology and Oncology, University of Kansas Medical Center, Kansas City, Kansas 66103

Received for publication July 20, 1973

M icrobodies (peroxisomes), the cytoplasmic other organized internal structure. The possibil-
constituents identified originally in kidney and ity that the structures identified recently as
liver, have only recently been recognized as microbodies in several cell types may contain
ubiquitous structures in animal cells (13, 14, 16, only catalase cannot be excluded.
20, 22, 31). De Duve and collaborators investi-
DETOXIFICATION OF HYDROGEN PEROXIDE
gated the biochemical composition of hepatic
microbodies and showed that these organelles De Duve and Baudhuin (9) discussed the
contain catalase and several oxidative enzymes various functional possibilities and suggested
(8, 9, 17, 23). It appears that catalase is the only that peroxisomes might play a role in gluconeo-
constant component of peroxisomes derived genesis or in the reoxidation of reduced nicotin-
from different sources, whereas the hydrogen amide adenine dinucleotide (NADH) produced
peroxide-producing oxidative enzymes that ac- in the cell sap, by the peroxisomal a-hydroxy
company catalase differ in number and in acid oxidase and catalase, which may provide
specificity (8). Identification of microbodies in additional electron shuttle systems. Because of
tissues other than liver and kidney was based on the association of hydrogen peroxide- producing
the cytochemical localization of catalase with oxidases and of catalase in hepatic microbodies,
the alkaline 3, 3’-diaminobenzidine medium de- it was also suggested that the peroxisomes play
scribed by Novikoff and Goldfischer (19). It a protective role against increased concentra-
remains to be determined whether these cata- tion of hydrogen peroxide in the cell (8, 9).
lase-containing cytoplasm ic structures contain However, it was pointed out that the protection
“at least one hydrogen peroxide producing oxi- offered by peroxisomes is only a partial one
dase” (8) in order to regard them as peroxi- because of the localization of peroxisomal en-
somes. zymes in individual particles (9).
Additional studies are necessary to character- Based on the morphologic studies in animals
ize the composition of these organelles derived treated with ethyl-a-chlorophenoxyisobutyrate
from different animal tissues for delineating (CPIB), Reddy and Svoboda (28, 34) suggested
their functional significance (32). The possibil- that hepatic microbodies are not unit or-
ity that “the enzyme composition of the micro- ganelles, but merely represent electron-opaque
bodies from different vertebrate cell types may peroxisomal enzymes circulating in the endo-
vary and that these organelles may perform plasmic reticulum (ER) channels. We have also
different functions in different cell types” was indicated that, for rapid detoxification of hydro-
invoked previously (32). Microbodies of normal gen peroxide and to serve as rapid transit
and neoplastic Leydig cells (Fig. 1) contain shuttle systems for transfer of electrons from
cylindrical structures in their matrix (32, 37) one part of the cell to another, the presence of
but most microbodies identified recently in circulating enzymes in ER channels is more
several other cells and tissues lack a nucleoid or effective than if the enzymes were concentrated
‘Studies described in this paper were supported in in individual particles (34). Since the micro-
part by U.S. Public Health Service Grants GM 15956 body enzymes are in ER channels, it is reasona-
and CA5680. ble to conclude that there is no catalase in the
2Presented at the Symposium on Peroxisomes
cell sap and that which is present in the
organized by The Histochemical Society on April 14,
1973, at its 24th Annual Meeting held in Atlantic supernatant fraction is due to rupture during
City, N.J. homogenization of ER channels accumulated
967
968 REDDY

--
.‘ . .#{149},-. ,..,- ...0

..j. . - , ... .1#{188}’’ :

- t1, .4

p’,. tL..
-d -
- #{149} .‘
.. ,. ,. 0 . :.. .

-I.. ‘;.. :- -.

.4. ,‘ ..‘ r

,. 0

t
1-. . . . --- .;s- :‘-,‘ .

FIG. Leydig1. (interstitial) cell tumor of rat testis induced by cadmium chloride. Microbodies (mb) reveal
diaminobenzidine reaction product. Tubular inclusions (arrows) are present in some microbody profiles.
x52,000.

with peroxisomal proteins (28). The differences composition of peroxisomes in different cell
in size of the isolated peroxisomal particles is types, but it is equally important to identify
attributed to varying amounts of microbody chemicals and drugs, as well as altered meta-
protein material enclosed by the ER mem- bolic states, that are capable of altering these
branes during the process of homogenization. structures (36).
The remainder that is not enclosed by the mem- Studies with ethyl-a-p-chlorophenoxyisobu-
branes appears in cell sap (28). The absence of tyrate have clearly established that this com-
cytochemically demonstrable catalase in the pound is valuable in investigating the biologic
hyaloplasm (24, 25) and the demonstration of an behavior of peroxisomes under different experi-
increased catalase activity in cell sap fractions mental conditions (12, 24,. 29, 30, 35, 41-43).
of CPIB-treated livers (15) are consistent with CPIB is a hypolipidemic drug which lowers
the conclusion that the catalase activity in cell serum triglycerides and cholesterol in man and
sap is due to release occurring during the proc- in experimental animals (3, 43). Marked in-
ess of cellular fractionation (15, 24). crease in number of hepatic microbody profiles
Since microbody profiles have been estab- occurs in male rats and in both sexes of acatala-
lished as ubiquitous structures in animal cells, semic mice following CPIB treatment (12, 24,
it may be reasonable to assume that the protec- 25, 41-43). The increase in number of microbody
tion against heavy concentration of H,O, is one profiles is associated with significant elevation
of the primary properties of these organelles. in catalase activity resulting from enhanced
synthesis of this enzyme (27). Because of the
MICROBODY PROLIFERATION AND temporal association of microbody increased
HYPOLIDEMIC DRUGS with elevated levels of hepatic catalase, the in-
crease in microbody formation was considered
In order to understand the importance of as an expression primarily of the increase in
microbodies in cellular metabolism, it is not catalase level (41). However, studies by Reddy
only relevant to determine the biochemical et at. (26) showed that when CPIB was ad-
 POSSIBLE PROPERTIES OF MICROBODIES (PER0xIS0MES) 969

ministered to rats receiving allylisopropylaceta- MICROBODIES: CHOLESTEROL AND STEROID

mide, an agent that blocks the synthesis of new SYNTHESIS

catalase, the increase in microbody number still The widespread occurrence of peroxisomes in
occurred even though the catalase activity in several cell types of vertebrates (1, 4, 5, 7, 11,
liver was negligible. The induction of microbody 14, 16, 18, 20, 22, 31, 32, 38) has given rise to
proliferation by CPIB in the absence of catalase additional speculation regarding the possible
synthesis suggests that CPIB might induce biologic properties of these organelles. Based on
the synthesis of yet unidentified proteins that the finding of microbodies in interstitial cells of
make up between one-half and two-thirds of the rat, mouse and guinea pig testis, Reddy and
total peroxisomal proteins (17). Svoboda (33) suggested that microbodies might
The biologic significance or the mechanism be related to synthesis, storage or utilization of
by which CPIB induces microbody proliferation cholesterol or androgens. It was pointed out that
or stimulates the synthesis of catalase and microbody catalase of tes4ticular interstitial cells
possibly other peroxisomal proteins is not fully could regulate the intracellular cholesterol pool
understood. It is, however, evident that the size and its utilization in androgen biosynthesis.
presence of large quantities of peroxisomal pro- Reddy and Svoboda (33) suggested that micro-
teins in liver of CPIB-treated animals is respon- bodies, possibly through the regulation of the
sible for the filling of numerous dilated ER NADPH levels, control androgen biosynthesis
channels and that the size, shape and number of and catabolism, since the testicular microsomal
microbody profiles reflect the amount of perox- hydroxylases as well as the 4-reductases are
isomal proteins present in the circulating pool NADPH-dependent (40). Isocitrate dehydro-
(28, 34). genase, a peroxisomal enzyme and glucose
Novikoff and Shin (21) suggested a possible 6-phosphate are considered as potential genera-
relationship of microbodies to lipid metabolism tors of NADPH in the testis. The possibility
and Caravaca et al. (6) showed that catalase that microbodies may be involved in storage
subunits affect the cholesterol levels. Because of and transport of cholesterol or androgens, in
significant increase in microbody population addition to participation in steroid biosynthesis,
and in catalase synthesis resulting from CPIB, a was also considered by us (33). Identical lines of
drug which has profound hypolipidemic proper- reasoning were offered recently by Black and
ties, a possible relationship between micro- Bogart (5) regarding the possible relationship of
bodies and lipid metabolism was invoked (41). peroxisomal enzymes to steroid biosynthesis in
The increase in microbody number and in the adrenal gland, which also contains numer-
catalase activity in the liver was believed to be ous peroxisomes (4, 18).
related to the lowering of serum cholesterol Microbodies were identified in spontaneous
levels by CPIB. However, additional studies and cadmium-induced interstitial cell (Leydig
have indicated that hepatic microbody prolifer- cell) tumors of rat testis (32, 37) and it appears
ation resulting from CPIB treatment may be that tumors capable of steroid synthesis have
independent of the hypolipidemic effect of the numerous microbodies. Detailed studies are in
drug (2, 26). Nevertheless, other evidence indi- progress on transplantable Leydig cell tumors of
cates a possible association between catalase rat testis, in an attempt to correlate the occur-
and cholesterol metabolism (6, 10, 36). Re- rence of microbodies with the production or
cently, Reddy, Svoboda and Azarnoff have nonproduction of steroid hormones.
shown that nafenopin, another hypohipidemic Petrik (22) and, subsequently, Schneeberger
compound, is capable of inducing significant (38, 39) described microbodies in Clara cells
increase in number of microbody profiles (36). and in granular pneumocytes in lungs. Regard-
This compound is structurally related to CPIB. ing the speculation that peroxisomes may be
The microbody proliferation (Fig. 2) and in- involved in cholesterol or steroid metabolism
crease in catalase synthesis resulting from nafe- (14, 33), it is of interest that granular pneumo-
nopin treatment were comparable to that in- cytes may be involved in the synthesis of
duced by CPIB. Additional studies are in prog- surfactant which is rich in cholesterol. The
ress to delineate the relationship between he- finding of large numbers of peroxisomes in the
patic microbody proliferation and drugs which duodenum, jejunum and ileum of guinea pigs by
lower serum cholesterol and triglyerides. Novikoff and Novikoff (20) also strengthens the
970 REDDY

FIG. 2. Female wild (Cs strain) mouse given nafenopin (0.125% in the diet) for 4 weeks. The liver cell reveals
numerous microbody (mb) profiles some of which contain nucleoids (n). Marked variation in size (arrows) and
shape of microbody profiles is evident. x 23,800.

possible relationship of microbodies to lipids, 2. Azarnoff D, Svoboda D: Microbodies in experi-

since absorption and transport of lipid is one of mentally altered cells. VI. Thyroxine displace-
ment from plasma proteins and clofibrate effect.
the important functions of absorptive cells of
Arch Int Pharmacodyn Ther 181:386, 1969
the small intestine. The presence of microbodies 3. Azarnoff D, Tucker D, Barr G: Studies with ethyl
in brown adipose tissue (1) and in Schwann chlorophenoxyisobutyrate (clofibrate). Metabo-
cells of dorsal root ganghia (7) also suggests that lism 14:959, 1965

microbody enzymes may play a role in lipid 4. Beard ME: Identification of peroxisomes in the
rat adrenal cortex. J Histochem Cytochem
metabolism.
20:173, 1972
In summary, the precise role of animal micro- 5. Black VH, Bogart BI: Peroxisomes in inner
bodies in cellular metabolism remains to be adrenocortical cells of fetal and adult guinea pigs.
elucidated. However, several studies suggest J Cell Biol 57:345, 1973
6. Caravaca J, Dimond EG, Sommers SC, Wenk R:
that microbodies may play a significant, but as
Prevention of induced atherosclerosis by peroxi-
yet unclarified, role in a variety of functions dase. Science 155:1284, 1967
such as gluconeogenesis, cholesterol and steroid 7. Citkowitz E, Holtzman E: Peroxisomes in dorsal
metabolism and detoxification of hydrogen per- root ganghia. J Histochem Cytochem 21:34, 1973

oxide. The possibility that microbodies may 8. de Duve C: Evolution of thyperoxisome. Ann NY
Acad Sci 168:369, 1969
have different functions in different tissues is to
9. de Duve C, Baudhuin P: Peroxisomes (micro-
be considered, although their role in lipid me- bodies and related particles). Physiol Rev 46:323,
tabolism and in detoxification of H2O2 may be 1966
considered universal. 10. Goldfischer 5, Roheim PS, Etlelstein D, Essner E:
Hypolipidemia in a mutant strain of “acatala-
semic” mice. Science 173:65, 1971
REFERENCES
11. Hand A: Morphologic and cytochemical identifi-
1. Ahlabo I, Barnard T: Observations on peroxi- cation of peroxisomes in the rat parotid and other
somes in brown adipose tissue of the rat. J exocrine glands. J Histochem Cytochem 21:131,
Histochem Cytochem 19:670, 1971 1973
 POSSIBLE PROPERTIES OF MICROBODIES (PER0xI80MEs) 971

12. Hess R, Stubhi W, Riess W: Nature of the chlorophenoxyisobutyrate. Biochem Biophys Res
hepatomegahic effect produced by ethyl-chloro- Commun 43:318, 1971
phenoxyisobutyrate in the rat. Nature (Lend) 28. Reddy J, Svoboda D: Microbodies in experimen-
208:856, 1965 tally altered cells. VIII. Continuities between
13. Hruban Z, Rechcigl M Jr: Microbodies and re- microbodies and their possible biologic signifi-
lated particles. Morphology, biochemistry and cance. Lab Invest 24:74, 1971
physiology. Int Rev Cytol, suppl 1, 20, 1969 29. Reddy J, Svoboda D: Ethyl-a-p-chlorophenox-
14. Hruban Z, Vigil E, Slesers A, Hopkins E: Micro- yisobutyrate induced hepatic microbody prolifera-
bodies. Constituent organelles of animal cells. tion in rat liver and ubiquinone concentration
Lab Invest 27:184, 1972 Experientia 27:1059, 1971
15. Krishnakantha TP, Kurup CKR: Increase in 30. Reddy J, Svoboda D: Proliferation of microbodies
hepatic catalase and glycerol phosphate dehydro- and synthesis of catalase in rat liver. Induction in
genase activities on administration of clofibrate tumor-bearing host by CPIB. Am J Pathol 73:99,
and clofenapate to the rat. Biochem J 130:167, 1971
1972 31. Reddy J, Svoboda D: Microbodies (peroxisomes):
16. Kuhn C: Particles resembling microbodies in identification in interstitial cells of the testis. J
normal and neoplastic perianal glands of dogs. Z Histochem Cytochem 20:140, 1972
Zellforsch Mikrosk Anat 90:554, 1968 32. Reddy J, Svoboda D: Microbodies in Leydig cell
17. Leighton F, Poole B, Lazarow P. de Duve C: The tumors of rat testis. J Histochem Cytochem
synthesis and turnover of rat liver peroxisomes. I. 20:793, 1972
Fractionation of peroxisome proteins. J Cell Biol 33. Reddy J, Svoboda D: Microbodies (peroxisomes)
41:521, 1969 in the interstitial cells of rodent testes. Lab Invest
18. Magalhaes MM, Magalhaes MC: Microbodies 26:657, 1972
(peroxisomes) in rat adrenal cortex. J Ultrastruct 34. Reddy J, Svoboda D: Further evidence to suggest
Res 37:563, 1971 that microbodies do not exist as individual enti-
19. Novikoff AB, Goldfischer 5: Visualization of per- ties. Am J Pathol 70:421, 1973
oxisomes (microbodies) and mitochondria with 35. Reddy J, Svoboda D: Microbody (peroxisome)
diaminobenzidine. J Histochem Cytochem matrix: transformation into tubular structures.
17:675, 1969 Virchows Arch [Zellpathol], in press
20. Novikoff PM, Novikof’f AB: Peroxisomes in ab- 36. Reddy J, Svoboda D, Azarnoff D: Microbody
sorptive cells of mammalian small intestine. J proliferation in liver induced by nafenopin, a new
Cell Biol 53:532, 1972 hypohipidemic drug: comparison with CPIB. Bio-
21. Novikoff A, Shin WY: The endoplasmic reticu- chem Biophys Res Commun 42:537, 1973
lum in the Golgi zone and its relations to micro- 37. Reddy J, Svoboda D, Azarnoff D, Dawar R:
bodies, Golgi apparatus and autophagic vacuoles Cadmium-induced Leydig cell tumors of rat tes-
in rat liver cells. J Microsc 3:187, 1964 tis: morphological and cytochemical study. J Nati
22. Petrik P: Fine structural identification of peroxi- Cancer Inst, in press
somes in mouse and rat bronchiolar and alveolar 38. Schneeberger EE: A comparative cytochemical
epithehium. J Histochem Cytochem 19:339, 1971 study of microbodies (peroxisomes) in great al-
23. Poole B, Higashi T, de Duve C: The synthesis and veolar cells of rodents, rabbit and monkey. J
turnover of rat liver peroxisomes. III. The size Histochem Cytochem 20:180, 1972
distribution of peroxisomes and the incorporation 39. Schneeberger EE: Development of peroxisomes in
of new catalase. J Cell Biol 45:408, 1970 granular pneumocytes during pre- and postnatal
24. Reddy J, Bunyaratvej 5, Svoboda D: Microbodies growth. Lab Invest 27:581, 1972
in experimentally altered cells. IV. Acatalasemic 40. Styhianou M, Forchielhi E, Tummillo M, Dorfman
(Csb) mice treated with CPIB. J Cell Biol 42:587, R: I. Metabolism in vitro of 4-C ‘4-testosterone by
1969 a human liver homogenate. J Biol Chem 236:692,
25. Reddy J, Bunyaratvej S, Svoboda D: Microbodies 1964
in experimentally altered cells. V. Histochemical 41. Svoboda D, AzarnofI D: Response of hepatic
and cytochemical studies on the livers of rats and microbodies to a hypohipidemic agent, ethyl chlo-
acatalasemic mice treated with CPIB. Am J rophenoxyisobutyrate (CPIB). J Cell Biol 30:442,
Pathol 56:351, 1969 1966
26. Reddy J, Chiga M, Bunyaratvej 5, Svoboda D: 42. Svoboda D, Azarnoff D, Reddy J: Microbodies in
Microbodies in experimentally altered cells. VII. experimentally altered cells. II. The relationship
CPIB-induced hepatic microbody proliferation in of microbody proliferation to endocrine glands. J
the absence of significant catalase synthesis. J Cell Biol 40:734, 1969
Cell Biol 44:226, 1970 43. Svoboda D, Grady H, Azarnoff D: Microbodies in
27. Reddy J, Chiga M, Svoboda D: Stimulation of experimentally altered cells. J Cell Biol 35:127,
liver catalase synthesis in rats by ethyl-a-p- 1967