ANDROLOGIA
31, 83-90 (1999) ACCEPTED: 16, 1998
by
of
L. Samanta, A. Roy and G. B. N. Chainy
Biochemistry Unit, Department of Zoology, Utkal University, Bhubaneswar, India
Key words. Testis-hexachlorocyclohexane-lipid peroxidation-antioxidant enzymes-ATPase-spermatogenesis
Summary. Age-related changes in rat testicular oxidative stress parameters were investigated. A biphasic pattern was evident for lipid peroxidation and for the activity ratio of superoxide dismutase to cdtalase and glutathione peroxidase with increasing age. In the first phase of life (birth-
7 days), a linear fall in lipid peroxidation was accompanied by a gradual increase in the enzyme ratio which was reversed in the second phase
(1 5-600 days). Glutathione and ascorbic acid levels increased from birth to the 45th day and remained unchanged up till 365days and then reduced at 600 days of age. The maximum level of H , 0 2 observed at birth gradually decreased till
90 days and remained unchanged up till 365 days of age; thereafter its level was elevated on day
600. The results suggest that an antioxidant defence system plays a crucial role in development and maturation of the rat testis. When the rats were treated with hexachlorocyclohexane during critical stages of testicular development (6th-30th day) and responses were evaluated on the 46th day of age, elevations in the levels of testicular lipid peroxidation and H 2 0 2 in levels of superoxide dismutase, catalase and ascorbic acid were observed. However, no change in glutathione and its metabolizing enzymes was recorded. O n the other hand, hexachlorocyclohex- ane elevated total testicular Ca2 +-Mg2 +-ATPase activity. The results advocate for impairment of testicular functions in adult age as a consequence of some permanent lesions induced by hexachloro- cycloliexane during critical stages of sexual maturation.
Correspondence: Prof. G. B. N. Chainy, Biochemistry Unit,
Department of Zoology, Utkal University, Bhubaneswar-
75 1 004, India.
U S . Copyright Clearance Center Code Statement: 0303-4569/99/3 102-0083 $14.00/0
Introduction
The developing testis is distinctively different from that of the mature one, because extensive morpho- logical, physiological and biochemical changes take place during sexual maturation. Recently,
Peltola et al. (1992) have described the develop- mental profiles of rat testicular antioxidant enzymes from 6 to 240days of age. However, information on changes in isoenzymes of superox- ide dismutase (SOD) and glutathione peroxidase
(GPx) along with lipid peroxidation (LPX) in the testis with respect to development and ageing is still needed. In the present study, developmental profiles of antioxidant enzymes, two nonenzymatic antioxidants (reduced glutathione and ascorbic acid), LPX and H 2 0 2 birth to 600 days of age were estimated to con- struct a composite picture of dynamic changes taking place in antioxidant profile of the testis during sexual maturation and ageing.
Earlier it was reported that hexachlorocyclohex- ane (HCH), an organochlorine pesticide, induces oxidative stress in the testis and the magnitude of induction is higher in the case of immature rats than the mature ones (Samanta & Chainy, 1997).
Similarly, alteration in the mitochondrial ATPases of the rat testis to HCH is more pronounced in immature rats than their mature counterparts (Roy
& Chainy, 1996). In the present study, in order to understand the impact of HCH exposure during critical stages of testicular development, rats were exposed to H C H during critical stages of sexual maturation (from 6 to 30 days of age) and changes in oxidative stress parameters and mitochondrial
Ca2 +-Mg*+-ATPases in the testis were recorded on the 46th day (onset of reproductive capacity) of age.

84 L. SAMANTA
AL.
Materials and methods
Animals and pesticide treatment
For the study of age-related changes in oxidative stress parameters, testes were removed after decapitation from 0-, 3-, 7-, 15-, 21-, 30-, 45-, 90-,
365- and 600-day-old rats (n=5). The testes of rats below the 30-day-old age groups were pooled and five such pools were made from each age group. To stud), the effect of HCH during critical stages of sexual maturation, 6-day-old rats (onset of spermatogenesis) weighing 6 g were divided into two groups. Animals of Group-I1 were adminis- tered HCH (i.p., 20 mg in 0.1 ml refined ground- nut oil per kg body weight per day) till 30 days of age (after the completion of the first meiotic cycle), while animals of Group-I served as a control and received an equal volume of the vehicle solution only. Animals kom each group were sacrificed on the 46th day (onset of reproductive capacity) of age by decapitation ( n = studied) and the testes were quickly dissected out, cleaned in normal saline (0.9%, w/v) solution and processed immediately for estimation of various oxidative stress parameters (Samanta & Chainy,
1997). Testicular mitochondria1 Ca2+-Mgz+-
ATPase activity was estimated as described earlier
(Roy & Chainy, 1996).
Chemicals
Technical HCH was obtained from Southern
Pesticide Corporation Ltd, India (CAS no. 58-89-8; y-isomer content = 23% by mass).
A l other chemicals were of the highest purified grade available.
Statistics
For the age-related study, one-way analysis of variance
(ANOVA) followed by Duncan’s New
Multiple Range Test was used. To find the level of significance between control and HCH-treated groups, the Student’s t-test was applied. A differ- ence was considered significant at a
Results
1 Age-related changes
Lipid peroxidation and H 2 0 2 content. Endogenous
LPX was very high at the time of birth. Its value declined significantly on the 3rd day of age and remained unaltered up till 365 days of age. Again, a marked increase was noticed on the 600th day of age. On the other hand, in vitro stimulation of
LPX by FeSO, and ascorbic acid was minimal on the day of birth, gradually increased till 21 days of age and thereafter no change was observed till
600 days of age (Figure 1A). The level of LPX in the nuclear and mitochondrial fractions did not change with respect to age till 365 days of age, but a significant increase was recorded in its level in both the fractions at the age of 600days
(Figure 1B). LPX in the microsomal fraction of the testis was lower in immature and young rats, and reached its maximum at the age of 90 days; thereafter, its level remained unaltered till 600 days of age (Figure 1B).
The level of H,O, in the testis of the rat was higher at the time of birth, decreased gradually till 90 days of age and remained unchanged up to
365 days, and thereafter its level increased signifi- cantly on the 600th day of age (Figure 1C).
Activities oj- antioxidant enzymes. O n day 0, activities of testicular CN--sensitive and CN--resistant
SODS were low and gradually increased and reached to the maximum value by 7 days of age.
Thereafter, their activities gradually decreased to one third of their value by 30 days of age and did not change till the age of 365 days. The activities of both the isoenzymes decreased further at the age of 600 days (Figure 2A).
Catalase (CAT) activity increased linearly from birth to the age of 30days and remained unchanged thereafter till 365 days of age.
However, a significant decrease in its activity was recorded on the 600th day of age (Figure 2B).
Testicular GPxs (total, Selenium-dependent and non-Selenium-dependent) activities decreased gradually from birth till 21 days of age. An elev- ation in their activities were recorded on the 45th day of age and remained unchanged till 365 days of age. However, a sharp rise in their activities was noticed at the age of 600 days (Figure 3A).
The activity of GR was low at the time of birth, increased linearly till 15 days of age and there- after, remained unaltered till 600 days of age
(Figure 3B).
Level of antioxidant small molecules. GSH content of the testis increased gradually till 30days of age and did not change much thereafter till 45 days of age. A sharp rise in its level was recorded on day
90 which remained unchanged till 365 days of age.
In 600-day-old rats, its level decreased to the level of 45 day-old rats (Figure 4A).
Ascorbic acid content of the testis increased from birth to the 45th day of age. Thereafter, its level remained unchanged till 365days of age, but decreased significantly at 600 days of age
(Figure 4B).
ANDROLOGIA 31, 83-90 (1999)

- 4
- 0
Nudeor Froclion
-
Microromd Frocllon
HCH 85
10 20 30 4 0
Age (Days)
I
90 365 600
Figure 1. Age-related changes in the level of lipid peroxidation in (A) crude homogenate and (B) various subcellular fractions, and (C) H,O, content of rat testis. Data are expressed as meankSD observations. of 5
ANDROLOGIA 31, 83-90 (1999)

86 L. SAMANTA
AL.
3 0 0 o--o Tolol SOD
SOD
T cw-0 CN-rsslrlaiil SOD 60
A . o--a
I
0-4
Tot01 GPx
Non-So-dapendsnl
So-dspendsnl GPa
GPx
.
.
.
.
.
.
o
10 2 0 30 40
A g e ( D a y s )
S O 3 6 5 6 0 0
10 20 30 40
-*-
SO
Age (Days)
365 600
Figure 2. Age-relaled changes in the activities of (A) superoxide dismutases (SOD) arid (B) catalase in rat testis. Data are expressed as mean SD of 5 ol)servations.
2 Efects ofHCH
Efect on @id peroxidation and H 2 0 2 content
(Table 1). The endogenous- as well as FeS04. and ascorbic acid- stimulated LPX in the crude homo- genate of the tcstis was elevated in HCH-treated rats. Although the LPX level did not change in the nuclear fraction in HCH-treated rats in com- parison to control animals, its level increased significantly in mitochondrial and microsomal fractions. Similarly, testicular increased in thr: pesticide exposed group only.
Efect on antioxidant enqmes and mitochondria1 Ca2
'
Mg2+-AIPase ('Table 2). The activities of cytosolic
CN--resistant SOD and CAT decreased in the
HCH-treated group, whereas activities of cytosolic
CN--sensitive SOD, Se-dependent and non-Se-
'365
-
600
I
0 10 20 30 40
Age (Days1
I'
90
Figure 3. Age-related changes in activities of (A) glutathione per- oxidases (GPx) and (B) glutathione reductase in rat testis. Data are expressed as mean SD of 5 observations. dependent GPx and G R remained unaltered. increase in HCH-treated rats in comparison to their control group.
Efect on nonenzymatic antioxidants (Table 3). Ascorbic acid content depleted significantly in the HCH- treated group in comparison to control ones, whereas GSH content remained unaltered.
Efect on sperm production and testicular histology
(Table3). Presence of spermatozoa in the cauda epididymis of control rats was recorded. The number of spermatozoa were very few. No
ANDROLOGIA 31, 83-90 (1999)

TESTICULAR
AND
HCH
T7
Table 2. Effect of hexachlorocyclohexane (20 mg kg-' body weight/day, i.p.) during critical stages of sexual matu- ration on activities of testicular antioxidant enzymes of rat.
Data are expressed as mean f
I
1
Enzymes Control Experimental
Total SOD
CN--sensitive SOD
CN--resistant SOD
CAT
Total GPx
Se-dependent GPx
Non-Se-dependent GPx
GR
CaZ+-Mg2+-ATPase
86.68 f
41.48 f
45.20f7.62
9.59 f0.67
73.67 f
16.75k 1.49
56.91 f
15.31 f0.50
31.10 f0.88
73.76 f4.48*
39.19f 1.56
34.57 f
7.70f 1.10*
73.71 k3.36
17.23f2.02
56.48 f
15.40f0.45
40.31 f0.97*
SOD: superoxide dismutase (units mg- protein), CAT: catalase (pkat mg- protein), GPx: glutathione peroxidase and GR: glutathione reductase (nmol NADPH
Pi liberated mg- protein h-').
*
P< 0.05 in comparison to respective control. degenerated and the tubular lumen was filled with necrosed cells.
Age IDoysl
Fignre 4. Age-related changes in (A) Reduced Glutathione (GSH) and (B) Ascorbic acid content of rat testis. Data are expressed as mean f of 5 observations. spermatozoa were found in the cauda epididymis of HCiH-treated rats.
Histological analysis of the pesticide-treated rat testis revealed degenerative changes. Most of the seminiferous tubules as well as Leydig cells were atrophied, the germinal epithelium was
Discussion
In this work age-related tissue oxidative stress has been studied by estimating tissue peroxidation by the thiobarbituric acid (TBA) test. In spite of its low specificity, it is the most widely used test for tissue oxidative stress. Its main advantage is its capacity to detect many kinds products and intermediates. of peroxidation
To the best of our knowledge, reports on age-related changes in in uivo
LPX in the testis of rat are not available. No significant change was observed in the TBA-reac- tive substance (TBARS) level in various subcellular fractions of the testis from 3 to 365 days of age. It is interesting to note that susceptibility to peroxi- dation of polyunsaturated-fatty acids (as indicated by stimulation of TBARS formation by FeSO,
~~ ~ ~~ ~
Table 1. Effect of hexachlorocyclohexane (20 mg kg-' body weight/day, i.p.) during critical stages of sexual maturation on lipid peroxidation (nmol mg-
' protein) in various testicular subcellular fractions and testicular H,O, content (nmol g-
' tissue wet weight) of rat. Data are expressed as mean f
Experimental
~~
_____
Crude homogenate (endogenous)
Crude homogenate (+ FeS04 & ascorbic acid)
Nuclear fraction
Mitochondrial fraction
Microsomal fraction
H P ,
*P< in comparison to respective control.
_
Control
_ _ _ _
0.85 f0.07
12.76 k0.44
1.42 f
1.18 f
3.35 f
31 1.95 f
~
1.46 5 0.32*
15.84f 1.34*
1.64f0.41
1.84 .t 0.45*
4.80 f
450.90 & 37.29*
ANDROLOCIA 31, 83-90 (1999)

88 L. SAMANTA
Table 3. Effect of hexachlorocyclohexane (20 mg kg-' body weight/day, i.p.) during critical stages of sexual maturation on rat testicular reduced glutathione (nmol/mg protein), ascorbic acid @g/g tissue) and sperm production. Data are expressed as mean & SD of 5 observations
Control Experimental
Reduced glutathione
Ascorbic acid
Mature spermatozoa seen in the epididymis
52.19 k6.07
236.07 -t 26.56
5 out of 5 rats
44.31 k5.55
193.48-t 14.30*
0 out of 5 rats and ascorbic acid) increased gradually from birth till 21 days of age and remained unaltered till
600days of age. This may be due to qualitative and quantitative changes occurring in phospholi- pid and fatty acid compositions of the testis during maturation (Johnson, 1970). The amount of phos- pholipid and fatty acid composition are two of the major factors that control lipid peroxidation (Bus
& Gibson, 1979).
Changes in SOD, CAT and GPx activities in the testis during sexual maturation are in good agreement with the results of Peltola et al. (1992).
In the mammalian system there are two main types of SOD. One is Cu/Zn-SOD, which is predominately cytoplasmic in nature, and the other one is a Mn-dependent SOD, which is mitochondria1 in nature (Weisiger & Fridovich,
1973). The present study indicates 30-40% of cytosolic SOD activity to be CN--resistant SOD
(Mn-SOD) and 75-78% of total GPx activity in the testis to be non-Selenium-dependent.
When the ratio of SOD/(CAT +GPx) was plot- ted against age, a biphasic pattern was observed
(Figure 5). In the first phase (0-7 days of age), a linear regression analysis of the data demonstrates an increase in SOD/(CAT
+
GPx) ratio as a func- tion of age ( r = +0.95), concomitant with a decrease in LPX value ( r = -0.68), whereas in the second phase (1 5-600 days of age) a linear fall in the SOD/(CAT+GPx) ratio ( r = -0.79) along with an increase in the LPX value ( r = +0.81) with respect to age was observed. The increasing ratio of SOD/(CAT
+
GPx) in the testis during differentiation indicates gradual decline in O2
- radical formation and a gradual increase in the capacity of the testis to remove H 2 0 z . Such linear increase in the ratio of enzymes will gradually shift the tissue from pro-oxidative condition to normox- idative condition. The presence of high SOD and low CAT and GPx activities in the testis of the
7-day-old rat lends support to the hypothesis of
Oberley et al. (1981) that H 2 0 2 in the regulation of normal cell division. To support the above argument, we found the level of HzO, in the testicular crude homogenate in the following order: immature >young > adult.
Furthermore, it was observed that ascorbic acid (a potent scavenger of oxyradicals) was low in the testis of immature and young rats in comparison to the adults. Therefore, it is suggested that SOD in conjunction with CAT and GPx may be involved in regulating the cell divisions and differ- entiation in the testis during sexual maturation.
Pradeep et al. (1990) have reported the existence of an alternate pathway of testosterone biosynthesis inducible by luteinizing hormone and mediated by SOD and peroxidases. The drop in SOD activity (from the 7th to 30th day of age) parallels the replacement of foetal-type Leydig cells by adult-type cells (from 2 to 5 weeks of age) and the concomitant decline in testosterone production.
As no effort has been made in this study to separate cell types, any suggestion at this stage concerning cellular localization of these enzymes would be speculative. However, Bauche et al.
(1994) have shown that Sertoli and peritubular cells have more SOD and GSH-dependant enzyme activities associated with high GSH level, whereas the meiotic cells are characterized by higher SOD activity and GSH content with very low GSH-dependant enzyme activity. Leydig cells displayed high GSH content, moderate SOD and
GSH-related enzyme activity, except for GPx, which was very high. Therefore, the relatively lower activities of GSH-dependant enzymes (GPx and GR) and higher GSH content in the adult testis could be due to the increase in the meiotic cell population during maturation.
Earlier studies from our laboratory showed that acute single dose exposure of HCH-elevated LPX in testicular subcellular fractions (Samanta &
Chainy, 1997), and repeated administration of the pesticide for 30 days showed a cumulative effect
[unpubl. data], and the effect is more pronounced in immature (1 5-day-old) rats. In the present study, treating the rats chronically with HCH during the critical stages of sexual maturation and then with- drawing the pesticide after completion of the first meiotic cycle show that the damage caused to the nucleus is reversible in nature, whereas that of
ANDROLOGIA 31, 83-90 (1999)

m w
0
1.5 r
I r a
1 . 0
!2
-
0
E c 0 . 5
-
X n
(3
+ c
I-
I
4 n
?!
.-
.I- m a
CT) +
7 c
4
0
?! I
.-
.I- m a
C
2
TESTICULAR
SYSTEM AND lor y=0.78+0.66x, r = +0.95
HCH 89
I
X n
1
I I I I I I
0 100 300 500 700
Age (Days) Age (Days)
I.
# y = 1 . 3 5 - O . I O x , r = - 0 . 6 8 IU%, r = to.81
E
.c
1.4 a
.I-
2
P
3
-
I ..6
-
E c
.:.
*
. .
:
0 . 2
- o 1
I
1 I I I 0
0 2 4 6 8 10
Age (Days)
Figure 5. Activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), and the level of lipid peroxidation in the testis of rat during development and maturation. (i) Scatter-plot of SOD/CAT+GPx activity vs. age in the testis of rat. (ii) Scatter-plot of lipid peroxidation vs. age in the testis of rat. mitochondria and microsomes is a permanent one.
Mitochondria and microsomes are reported to play an important role in testicular steroidogenesis
(Tomaoki, 1973; Georgiou et al., 1987; Hall, 1994).
Therefore, disruption in their integrity will affect testicular function. Previously, it has also been demonstrated that these two organelles show more susceptibility to LPX than the nuclear fraction as a consequence of extraneous testosterone adminis- tration to adult intact rats (Chainy et al., 1997).
Furthcrmore, HCH is reported to diminish the activities of testicular steroidogenic enzymes (Pius et al., 1990). Therefore, the induction of oxidative stress in the testis appears to be due to a decline in iritratesticular testosterone concentration.
Delhumeau-Ongay et al. (1973) demonstrated that
ATP catabolism in testicular mitochondria of the rat takes place mainly due to the activity of Ca2+-
Mg2+-ATPase, and the enzyme activity also changes during maturation. The authors have also observed an augmentation in the enzyme activity concomitant with germinal cell regression induced
ANDROLOGIA 31, 83-90 (1999) either surgically or chemically. Therefore, an increase in Ca2+-Mg2+-ATPase activity in rat testicular mitochondria in response to HCH expo- sure during critical stages of sexual maturation may be due to degeneration of germ cells. In fact, routine histological analysis of testis of HCH- treated rats revealed an adverse impact on sperma- togenesis concomitant with absence of spermato- zoa in the epididymal smear of HCH-treated rats.
Results of the present study show a decline in the activities of testicular CN --resistant SOD and
CAT. This is in conformity with our previous findings (Samanta & Chainy, 1997). In cellular systems GPx and CAT are responsible for effective neutralization of H,O,. And intracellular H , 0 2 concentration is one of the factors in deciding which of these isoenzymes operate as both have a different Km value for the substrate, i.e. CAT:
25 mM (Lehninger et al., 1993) and GPx: 1-10
PM
(Barman, 1974). The reduced activity of SOD in response to H C H and the need for a high H 2 0 , level for operation of CAT together with the

90 L. SAMANTA
AL. lowered activity of GPx of w l lead to accumulation
H,O, in the testis. In fact, we observed an elevated level of H,O, in response to HCH. Such a high concentration of H 2 0 2 will have more chance of generating highly cytotoxic 'OH radicals by reacting with elevated response to HCH (Kuhn et
02' generated in al., 1986; Bagchi &
Stohs, 1993). The 'OH radical due to its high reactivity can attack almost all macromolecules of the cell and consequently, the protection from the enzymatic and nonenzymatic antioxidants in the testis will not be sufficient and lead to cell damage and death.
The results of the present study suggest that pesticide exposure to the testis during critical stages of sexual maturation produced some irreversible damage through induction of oxidative stress and this may cause testicular dysfunction in adulthood.
Acknowledgements
We thank the Head, Department of Zoology,
Utkal University for providing laboratory faci- lities. Financial assistance from University Grants
Commission, India and Council of Scientific and
Industrial Research, India is gratefully acknowledged.
References
Bagchi M, Stohs SJ (1993) In nitro induction of reactive oxygen species by 2,3,7,8-tetrachlorodibenzo-p-dioxin, lindane in rat peritoneal macrophages and hepatic mito- chondria and microsomes. Free Rad Biol Med 14:ll-18.
Barman TE (1971) Enzymes handbook, (Suppl. I) Springer-
Verlag, New York, pp. 136-137.
Bauche F, Fouchard M, Jegou B (1994) Antioxidant system in rat testicular cells. FEBS Letters 349:392-396.
BusJS, Gibson J E (1979) Lipid peroxidation and its role in toxicology. In: Reviews in Biochemical Toxicology.
Hodgson E, Bend JR, Phdpot R M (eds) Elsevier, North-
Holland, New-York, pp. 125-149.
Chainy GBN, Sainantaray S, Samanta L (1997) Testosterone- induced changes in testicular antioxidant system.
Andrologia 29:343-349.
Delhumeau-Ongay G, Trejo-Bayona R, Lara-Vivas L (1 973) in rat testis throughout maturation. J Reprod Fert
33:513-517.
Georgiou M, Perkins LM, Payne AH (1 987) Steroidsynthesis- dependent, oxygen-mediated damage of mitochondria1 and microsomal cytochrome P-450 enzymes in rat Leydig cell cultures. Endocrinology 12 1 : 1390-1 399.
Hall PF (1994) Testicular steroid synthesis: organiation and regulation. In: The Physiology of Reproduction, Vol 1.
Knobil E, Neil JD (eds) Raven Press, New York, pp. 1335-1362.
Johnson AD (1970) Testicular lipids. In: The Testis, Vol 2.
Johnson AD, Gomes WR, Vandermark NL (eds) Academic
Press, New York, London, pp. 193-258.
Kuhn DB, Kaplan SS, Basford RE (1986)
Hexachlorocyclohexanes, potent stimuli of 0, - production and calcium release in human polymorphonuclear leuco- cytes. Blood 68:535-540.
Lehninger AL, Nelson DL, Cox MM (1993) Principles of
Biochemistry. CBS Publishers & Distributors, Delhi,
216 pp.
Oberley LW, Oberley TD, Buettner GR (1981) Cell division in normal and transformed cells: the possible role of superoxide and hydrogen peroxide. Medical Hypothesis
7:21-42.
Peltola V, Huhtaniemi I, Ahotupa M (1992) Antioxidant enzyme activity in the maturing rat testis. J Androl
13~450-455.
Pius J, Shivanandappa T, Krishnakumari MK (1990)
Protective role of vitamin A in the male reproductive toxicity of hexachlorocyclohexane (HCH) in the rat.
Reprod Toxicol 4:325-330.
Pradeep KG, Seerwani N, Laloraya M, Nivsarkar M, Verma
S, Singh A (1990) Superoxide dismutase as a regulatory switch in mammalian testicular steroidogenesis. Biochem
Biophys Res Commun 173:302-308.
Roy A, Chainy GBN (1996) Age-related change in rat testicular ATPase activities in response to HCH treatment.
Bull Environ Contam Toxicol 56: 165- 1 70.
Samanta L, Chainy GBN (1 997) Comparison of hexachloro- cyclohexane-induced oxidative stress in the testis of imma- ture and adult rats. Comp Biochem Physiol 118C:319-327.
Tomaoki BI (1973) Steroidogenesis and cell structure.
Biomchemical pursuit of sites of steroid biosynthesis.
J Steroid Biochem 489-1 18.
Weisiger RA, Fridovich I (1973) Mitochondria1 superoxide dismutase. Site of synthesis and intramitochondrial localiz- ation. J Biol Chem 248:4793-4796.
ANDROLOGIA 31, 83-90 (1999)