Hormonal Regulation of Cytochrome P-450 - Cancer Research

(CANCER RESEARCH 40. 2486-2492. July 1980]

Hormonal Regulation of Cytochrome P-450-dependent MonooxygenaseActivity and Epoxide-metabolizing Enzyme Activities in Testis of

Hypophysectomized RatsInsu P. Lee,1 Katsushi Suzuki,2 Hasan Mukhtar, and John R. Bend

Laboratory of Reproductive and Developmental Toxicology Â¡I P. L.. K. S.]. and Laboratory of Pharmacology Â¡H.M.. J. R. B.J. National Institute of EnvironmentalHealth Sciences, Research Triangle Park. North Carolina 27709

ABSTRACT

Since the male gonad is a target organ of both pituitaryhormones and androgens, the present study was performed todetermine whether testicular aryl hydrocarbon hydroxylase(AHH), epoxide hydrase (EH), and glutathione transferase activities and cytochrome P-450 levels were regulated by follicle-

stimulating hormone (FSH), luteinizing hormone (LH), and testosterone. Intact, sham-operated controls and hypophysecto-

mized (HYPOX) male rats were used. Forty days after HYPOX,rats received three daily injections of LH, FSH, testosterone,orLH plus FSH (100 fig each), or 0.15 M NaCI. Rats were killed5, 10, and 15 days after hormone administration. In HYPOXrats, testicular AHH and EH activities and P-450 content weresignificantly reduced at all time points (20 to 70% of sham-

operated control values), but glutathione transferase activitywas unchanged. In contrast, testicular AHH, EH, and P-450

were significantly induced in all LH treatment groups but not inFSH or testosterone treatment groups. The highest inductionwith LH was found in the 15-day treatment group (5- to 14-foldthat of 0.15 M NaCI-treated HYPOX control). Since Leydig cellsare target cells of LH, the result suggests that LH-stimulatedinduction of testicular AHH and EH activities and P-450 contents may be primarily associated with the interstitial cell compartment of the testis.

INTRODUCTION

Pollution of the environment has increased over the pastdecades to the point where human exposure to chemicalcontaminants is almost inevitable. All organs and tissues arepotential targets for chemical toxicity, with the testis being oneof the more critical since testicular exposure to certain pollutants might increase the frequency of germ cell mutations andconsequently be associated with genetically linked disease.

The incidence of xenobiotic-induced germ cell damage andsterility appears to be increasing. Many organic solvents (7),inorganic metals (19, 22), drugs (10, 20), pesticides (26), andPAH3 (1, 9, 28) are toxic to male and female reproductive

organs in various species. Such findings warrant more intensive

' To whom requests for reprints should be addressed, at the Laboratory of

Reproductive and Developmental Toxicology. National Institute of EnvironmentalHealth Sciences, P. O. Box 12233. Research Triangle Park. N. C. 27709.

' Present address: Department of Physiology, Nippon Veterinary and Zo-

otechnical College, Tokyo. Japan.1The abbreviations used are: PAH. polycyclic aromatic hydrocarbons; EH.

epoxide hydrase; AHH. aryl hydrocarbon hydroxylase; GSH-T. glutathione transferase; FSH, follicle-stimulating hormone; LH, luteinizing hormone; 4,5-BPO,benzo(a)pyrene 4.5-oxide; BP, benzo(a)pyrene; HEPES, fV-2-hydroxyethylpiper-azine-A/'-2-ethanesulfonic acid.

Received January 11,1980; accepted April 15. 1980.

examination of the effects of various classes of chemicals,including pollutants, on germ cells.

With respect to PAH-mediated pathology, it is now generally

accepted that the mutagenic and carcinogenic effects of PAHare associated with metabolic activation to electrophilic compounds such as arene oxide or diol oxide derivatives. Reactiveepoxides formed by cytochrome P-450-dependent monooxy-

genase activity can interact with cellular nucleophiles (11, 12,18), undergo further biotransformation (catalyzed by EH) todihydrodiols, rearrange nonenzymatically to phenols, or reactenzymatically (catalyzed by the glutathione transferases) andnonenzymatically with glutathione (3, 16). Interaction with glutathione generally results in detoxication. With many areneoxides, hydration results in detoxication but with others, including benzo(a)pyrene 7,8-oxide, hydration is required to formbenzo(a)pyrene 7,8-dihydrodiol, the metabolic precursor ofbenzo(a)pyrene 7,8-dihydrodiol-9,10-epoxide, an ultimate car

cinogenic and mutagenic form of benzo(a)pyrene. In this example, EH could be considered as a metabolic activation step(2, 39, 42).

Although the hepatic cytochrome P-450-dependent mono-

oxygenase system is the major site for biotransformation ofmost foreign chemicals, testicular and ovarian microsomalmonooxygenase systems also are capable of activating anddetoxifying environmental chemicals (31, 32). The presence ofcytochrome P-450-dependent steroid-metabolizing enzymes(38), the postnatal development of epoxide-metabolizing en

zymes in the testis (31) and ovary (32), and the differentialdistribution of AHH, EH, and GSH-T activities in spermatogenic

and interstitial cell compartments of rat testis (31) have beenstudied previously. We showed that specific activities of epoxide-metabolizing enzymes were very low in prepubertal testis

but increased markedly at puberty concomitant with significantincreases in plasma levels of FSH, LH, and testosterone (31).

AHH, EH, and GSH-T activities in interstitial and germ cell

compartments of the mammalian testis may be a contributingfactor to germ cell toxicity, or lack thereof, following exposureto pollutant chemicals that are biotransformed to reactive metabolites. The present study was performed to determinewhether AHH, EH, and cytosolic GSH-T activities of rat testis

are regulated by FSH, LH, or testosterone.

MATERIALS AND METHODS

Chemicals and Hormones. [3H]-4,5-BPO (specific activity,

10 mCi/mmol; radiochemical purity, >99%) and unlabeled 4,5-

BPO were synthesized by Midwest Research Institute, KansasCity, Mo. on National Cancer Institute Contract 1-CD-33387

supplemented by National Institute of Environmental Health

2486 CANCER RESEARCH VOL. 40

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Hormonal Regulation of Gonadal PAH-metabolizing Enzymes

Science. BP was obtained from the Eastman Kodak OrganicChemical Company, Rochester, N. Y. HEPES, glucose 6-phos-phate, glucose-6-phosphate dehydrogenase (type XI), and

NADP were purchased from Sigma Chemical Company, St.Louis, Mo. Other chemicals were of reagent grade and wereobtained from standard commercial sources. FSH (NIH-ovine-

. FSH-S-12) and LH (NIH-ovine LH-S-20) were provided by the

Endocrinology Study Section, National Institute of Arthritis,Metabolism, and Digestive Diseases, NIH, Bethesda, Md.; themean relative potencies of FSH and LH were 1.02 to 1.33units/g and 0.89 to 1.24 units/mg. Testosterone propionatewas obtained from Sigma.

Animals and Experimental Design. To study the effects ofFSH, LH, and testosterone administration on cytochrome P-450 content and specific AHH, EH, and GSH-T activities oftestis and liver, control adult male sham-operated and hypoph-ysectomized Sprague-Dawley rats (10 weeks old) were ob

tained from Charles River Breeding Laboratories, Inc., Wilmington, Mass., and used throughout the experiments. Animalswere housed 3/cage and allowed free access to a rodent diet(NIH-31S) and drinking water containing 5% dextrose for the

first postoperative week. Animals were maintained under stableconditions of illumination (14 hr light, 10 hr dark cycle) andtemperature (25Â°).The experimental groups consisted of con

trol, sham-operated, and hypophysectomized rats. Each group

consisted of 60 male rats with the exception of the hypophysectomized animals (240 male rats) which were subdivided into4 groups. Four days after surgery, each of the hypophysectomized groups received 3 daily s.c. injections (9 a.m., 1 p.m.,and 5 p.m. daily) of either FSH (100 Â¿ig),LH (100 /ig), or FSHplus LH (100 /Â¿geach) in 0.1 ml of 0.15 M NaCI per injectionper rat or testosterone (100 fig) in 0.1 ml of corn oil perinjection per rat. Groups of control and sham-operated ratswere included in each treatment group and they received 0.1-ml s.c. injections of either 0.15 M NaCI or corn oil (3 timesdaily).

Preparation of Microsomes and Cytosolic Fractions. Allanimals were killed by decapitation between 8 and 10 a.m. onDay 0, 5, 10, or 15 following hormone treatment. Testes wereimmediately excised, weighed, and homogenized with a Potter-Elvehjem homogenizer with a Teflon pestle (5 strokes of pestlefor testis) in 4 volumes of ice-cold 0.15 M KCI in 0.02 M HEPESbuffer at 4Â°,pH 7.4. The homogenate was centrifugad for 15min at 9000 x g at 4Â°.The mitochondrial supernatant was

centrifuged at 176,000 x g for 45 min to obtain microsomaland microsomal supernatant fractions. The microsomal pelletwas resuspended in KCI-HEPES buffer, washed once, andresuspended in KCI-HEPES with a small Potter-Elvehjem ho

mogenizer. The protein concentration of both microsomes andsupernatant fractions was determined according to the methodof Lowry ef al. (24) using bovine serum albumin (Fraction IV)as the standard.

AHH Assays. Microsomal AHH activity was determined by amodification of the method of Wattenberg ef al. (41). Theincubation mixture (1 ml total volume) contained 8.7 mM glucose 6-phosphate, 8.7 mM MgSO4, 2.2 mM NADP*, 1.7 units

of glucose-6-phosphate dehydrogenase, 1.2 mg of bovine

serum albumin (fraction IV), 0.17 M HEPES buffer (pH 7.4),and 0.05 ml microsomal fraction (0.5 to 1.5 mg protein perassay) and was incubated at 37Â°in a Dubnoff metabolic shaker.

The reaction was initiated by the addition of BP in 10 jul of

acetone to yield a final concentration of 0.1 mM. Incubationwas for 30 min for testicular assays. The reaction was terminated by the addition of 4 ml acetoneihexane (1:3, v/v),followed by thorough extraction and centrifugation at 700 X gfor 7 min. An aliquot of the organic phase (2.5 ml) was transferred to another tube. An equal volume of 1 N NaOH wasadded, and the mixture was shaken vigorously and centrifugedagain at 700 x g for 7 min. The fluorescence in the aqueouslayer was determined at an excitation wavelength of 392 nmand an emission wavelength of 522 nm. The quantitation ofphenolic BP metabolites was based on comparison of fluorescence to a 3-hydroxybenzo(a)pyrene standard.

EH and GSH-T Assays. EH and GSH-T activities were de

termined in microsomal and 176,000 x g supernatant fractionsof the testes, respectively. The assays for EH and GSH-Tactivities were performed with [3H]-4,5-BPO as the substrate.

The products [benzo(a)pyrene-4,5-dihydrodiol for EH and theglutathione conjugate of 4,5-BPO for GSH-T] were quantitatedradiometrically (3, 16, 17, 25). All assays were corrected fornonenzymatic product formation.

Spectral Analysis. Cytochrome P-450 was assayed accord

ing to the method of Omura and Sato (34). An extinctioncoefficient of 91 mM"' cm"' was assayed for the quantitation

of P-450. The microsomal protein concentration ranged from

2 to 5 mg/ml in 0.17 M HEPES buffer, pH 7.4. Spectra wererecorded with an Aminco DW-2 dual-beam recording spectro-

photometer (American Instrument Company, Silver Spring,Md.).

Morphological Studies. Whole testes from 3 rats of eachtreatment group were fixed in Bouin's fixative for 18 hr and

then washed in distilled water followed by repeated washing in70% ethanol before dehydrating in 100% ethanol. Testiculartissue was imbedded in JB-4 media, sectioned at 2 /nm thick

ness, and stained with hematoxylin and eosin prior to microscopic examination.

Statistical Analysis. Statistical comparisons were made using the Student's f test (40), and p < 0.01 was taken to

represent significant differences between means.

RESULTS

Characteristics of Testicular Microsomal AHH. Preliminaryexperiments were first performed to elucidate optimal AHHassay conditions in rat testicular microsomes. Chart 1 showsthe effect of pH, microsomal protein concentration, and incubation time on AHH activity in testicular microsomes. The pHoptimum for testicular AHH activity was broad between 7.2 and7.8 and differed from that of hepatic AHH activity which wasbetween 7.0 and 7.4 (data not shown). The rate of 3-hydroxy-

benzo(a)pyrene formation was linear over the range of microsomal protein normally utilized [3 mg per incubation for testisand 1.5 mg for liver (data not shown)]. The reaction rates fortesticular and hepatic AHH enzyme activities were linear for 30and 10 min (data not shown), respectively.

Response of Rats to Hormonal Treatment. Relative to eitherintact control rats or sham-operated control rats, body and

testicular weights of hypophysectomized male rats were significantly decreased to 21 7 Â±22.9 (S.D.) and 0.60 Â±0.03 g,respectively (56%, and 17% of values for intact control rats at70 days of age). As shown in Table 1, hormone treatments didnot significantly change body or liver weights of hypophysec-

JULY 1980 2487



/. P. Lee et al.

tomized rats receiving FSH, LH, FSH plus LH, or testosterone,relative to hypophysectomized rats treated with 0.15 M NaCI.By contrast, the weights of the testes were significantly increased in response to all hormone treatments (p < 0.01).FSH plus LH treatment was the most effective in increasingtesticular weight; testicular weight 15 days after treatment ofhypophysectomized rats with FSH and LH was 3 times that ofhypophysectomized controls, but still only one-half of eitherintact or sham-operated control animals.

Effect of Hypophysectomy on Testicular AHH, EH, andGSH-T Activities and Cytochrome P-450 Content. Testicular

-20IIlÃ¬I o< a

= 1 ,0

li

ilII7O 74

PH78 5 IO 2O 30

Incubation time(min)

Chart 1. Effects of pH. microsomal enzyme concentration (cone.), and varyingincubation time. The assay condition of AHH activity of testicular (â€¢)was thatdescribed in "Materials and Methods" with the exception of pH of the incubation

media, enzyme concentration, and the incubation time. The microsomes(176,000 x g) from each group of experiments were prepared from 9 animals.Points, means of 3 to 5 experiments; oars, S.D.

AHH, EH, and GSH-T activities and cytochrome P-450 contentof intact control, sham-operated control, and hypophysecto

mized male rats 40 days after surgery are presented in Table2. Testicular microsomal AHH and EH activities and cytochrome P-450 content were significantly lower in hypophysectomized male rats than in those of intact or sham-operated

male rat controls. The specific testicular AHH and EH activitiesand cytochrome P-450 content in hypophysectomized rat

testes were about 60, 40, and 40%, respectively, of intact andsham-operated controls (based on the mean values). However,testicular glutathione S-transferase activity (with 4,5-BPO as

the substrate) was unchanged in hypophysectomized animals.AHH Activity following Hormone Treatment. The effects of

administration of the pituitary hormones, LH and FSH, andtestosterone to hypophysectomized rats on testicular AHHactivity were also studied. Testicular microsomal AHH activityof male hypophysectomized rats treated continuously with LHor with LH plus FSH for 5, 10, or 15 days after surgery wassignificantly greater than in hypophysectomized rats treatedwith 0.15 M NaCI (Table 3).

When administered to hypophysectomized rats for 15 days,LH increased testicular AHH activity about 14-fold, whereasFSH plus LH increased AHH activity only about 7-fold. How

ever, the diminution of AHH activity with time in the 0.15 MNaCI-treated hypophysectomized controls was the major con

tributing factor to the magnitude of the changes in AHH activityafter 15 days of hormone administration (at 10 days of treatment, testicular AHH activity was increased about 4-fold in LH-treated animals and about 2.5-fold in those receiving FSH plus

Table 1

Effects of hormone administration on body and testes weights in hypophysectomized male rats

The body weight of all experimental groups was determined immediately before sacrifice, and liver andtesticular weights were determined just after sacrifice. Hypophysectomy was performed at 70 days of agefor all rats. Each treatment group received either 0.15 M NaCI or the designated hormone, 3 times daily s.c.as described in "Materials and Methods." Body liver and testicular weights for intact control rats (110 days

old) were 390 Â±21.2 (4), 15.65 Â±1.43 (4), and 3.46 Â±2.83 (8); sham-operated (110 days old) were402.5 Â±8.7 (4), 1.7 Â±1.78 (4), and 3.36 Â±1.02 (9); hypophysectomized control (110 days old) were21 7.4 Â±22.9 (12), 10.11 Â±0.35 (4). and 0.60 Â±0.03 (24), respectively.

Organ wt (g) at following treatmenttimesHormones0.1

5 M NaCIBodyLiverTestesFSHBodyLiver

TestesLHBodyLiver

TestesFSH

+LHBodyLiver

TestesTestosteroneBodyLiver

Testes5

days218.00

Â± 8a(5)Â°8.65Â± 0.20"(4)0.58

Â±0.05(18)220.00

Â± 6(5)7.82Â± 0.72 (4)

0.89 Â±0.06(15)Â°223.00

Â±17(5)8.22Â± 0.87 (4)

0.83 Â±0.04(15)Â°222.00

Â±14(5)9.07Â± 0.39 (4)

0.96 Â±0.05(15)Â°221.00

Â±26(5)7.98Â± 1.15 (4)

0.63 Â± 0.05(15)1

0days214.00

Â±12(5)8.52Â± 1.60(4)0.54Â± 0.02(14)219.00

Â±11(5)7.87Â± 0.41 (4)

1.07Â± 0.12(8)Â°233.00

Â±15(15)7.35Â± 0.89 (4)

0.91 Â± 0.04(10)c232.00

Â±20(5)8.19 Â± 0.16 (4)

1.28 Â± 0.06(9)Â°235.00

Â±20(5)8.19Â± 0.47 (4)

0.79 Â± 0.17 (9)1

5days227.00

Â±16(5)8.10Â± 1.10(4)0.52

Â± 0.03(23)228.00

Â±12(5)7.10Â± 0.84 (4)1.19Â± 0.21(11)c21

7.00 Â± 5(5)7.40Â± 0.69 (4)

0.97 Â±0.10(11)Â°222.00

Â±11(5)7.86Â± 0.17 (4)

1.46Â±0.08(11)Â°231

.00 Â±11(5)762 Â± 0.45 (4)

0.85 Â± 0.11 (11)c

a Mean Â±S.D. for all values.0 Numbers in parentheses, number of animals.Â°Significantly different from hypophysectomized controls; p < 0.01.





Table 2Testicular AHH. EH, and GSH-T activities and cytochrome P-450 content of intact control, sham-operated.

and hypophysectomized male ratsAssays were determined simultaneously with preparation from intact control, sham-operated, and

hypophysectomized rats 40 days after hypophysectomy or sham surgery. Four rats were used for eachexperiment.

ControlSham-operatedHypophysecto

mizedAHH

(pmol/min/mg microsomalprotein)1.31

Â±0.12a

1.46 Â±0.170.84 Â±0.32CEH

(nmol/min/mgmicrosomal protein)0.93

Â±0.040.88 Â±0.080.35 Â±0.05Â°GSH-T

(nmol/min/mg cytosol(protein)20.00

Â±1.1020.30 Â±1.0018.90 Â±0.89Cytochrome

P-450(nmol/mg micro

somalprotein)0.12

Â±0.010.12 Â±0.0040.05 Â±0.00e

Mean Â±S.D. for all values.h The mean values of testicular enzyme activities in hypophysectomized rats were obtained from pooled

testes (4 to 6 animals/preparation).0 Significantly less than control and sham-operated control values (p < 0.01 ).

Table 3

Effects of hormone administration on testicular microsomal AHH activity inhypophysectomized male rats

AHH enzyme activity was determined using benzo(a)pyrene as a substrate.For each AHH assay, testes from 4 to 6 hypophysectomized rats were pooled.

AHH activity (pmol/min/mg microsomal protein) at followingtreatment times

Hormones0.1

5 M NaCIFSHLHFSH +LHTestosterone5

days0.83Â±0.26a

1.25 Â±0.222.31 Â±0.44Â°1.84 Â±0.13C

1.33 Â±0.371

0days0.93

Â±0.181.05 Â±0.233.68 Â±0.78C2.38 Â±0.20C

0.75 Â±0.301

5days0.35Â±0.166

0.44 Â±0.08o4.72 Â±0.93Â°2.36 Â±0.24C0.28 Â±0.07fc

Mean Â±S.D. of 3 experiments for all values.6 Significantly less than the 0 time control (prior to hormone treatment); p <

0.01 (see Table 2 for 0 time control values).c Significantly greater than the 0.15 M NaCI-treated (hypophysectomized

control); p< 0.01.

LH). In any case, the data suggest that FSH antagonizes theLH-mediated increase of specific AHH activity in testicular

microsomes. Testicular AHH activity in hypophysectomizedmale rats receiving 0.15 M NaCI, FSH alone, or testosteroneshowed marked decreases in AHH activity between 10 and 15days of treatment (Table 3).

EH Activity in Hypophysectomized Rats following Hormone Treatment. The effects of administering various hormones on testicular microsomal EH activity in hypophysectomized rats were similar to those obtained with AHH activity(Table 4). Testicular microsomal EH activity was markedlyincreased by LH and, to a lesser extent, by LH and FSH in atime-dependent manner after 15 days of treatment. Specificenzyme activities were about 5-fold greater in rats treated withLH and about 4-fold greater in rats given both LH and FSH than

in hypophysectomized controls. Testicular microsomal EH activity in hypophysectomized rats was not influenced by administration of FSH or testosterone alone.

Microsomal Cytochrome P-450 Content in Hypophysecto

mized Rats following Hormone Treatment. As with AHH andEH activity, treatment of male hypophysectomized rats (10weeks old) with LH and with LH plus FSH caused a significantincrease in testicular microsomal cytochrome P-450 content;with LH there was almost a 10-fold increase and with LH andFSH the increase was approximately 9-fold (Table 5). Increasesin P-450 content were time and dose related with both hormone

treatments. In contrast, FSH and testosterone treatment hadno effect. For comparison, testicular cytochrome P-450 con

tent in microsomes from hypophysectomized male rats (125

Table 4

Effects of hormone administration on testicular microsomal EH activity inhypophysectomized male rats

EH activity was determined using [3H]benzo(a)pyrene 4.5-oxide as substrate.

For each EH assay, testes from 4 to 6 animals were pooled.

EH activity (nmol diol/min/mg microsomal protein) at following treatment times

Hormones0.1

5 M NaCIFSHLHFSH + LHTestosterone5

days0.38Â±0.08a

0.41 Â±0.110.92 Â±0.13b0.82 Â±0.026

0.56 Â±0.131

0days0.35

Â±0.050.45 Â±0.01.77 Â±0.07b1.53 Â±0.036

0.53 Â±0.171

5days0.54

Â±0.090 78 Â±0.172.84 Â±0.32o

2.29 Â±0.150.58 Â±0 04

Mean Â±S.D. of 3 experiments for all values.Significantly greater than the 0.15 M NaCI controls (hypophysectomized); p

Table 5Effects of the selected hormone administration on testicular cytochrome P-450

content in hypophysectomized male ratsTesticular cytochrome P-450 was measured in microsomes prepared from the

pooled testes of 4 to 6 rats.

Testicular cytochrome P-450 content (nmol cytochrome P-450/mg microsomal protein) at following treatment times

Hormones0.1

5 M NaCIFSHLHFSH + LHTestosterone5

days0.03Â±0.0Â°

0.05 Â±0.010.10 Â±0.01o0.10 Â±0.016

0.03 Â±0.011

0days0.02

Â±0.010.03 Â±0.010.21 Â±0.01 b0.15 Â±0.02Â°

0.02 Â±0.011

5days0.03

Â±0.00.03 Â±0.010.30 Â±0.02b0.28 Â±0.02Â°

0.03 Â±0.01a Mean Â±S.D. of 3 experiments for all values.b Significantly different from the 0.15 M NaCI controls (hypophysectomized);

p < 0.01.

days old; 55 days after surgery) was only about 30% of thevalues for intact or sham-operated control rats of the same

age.Cytosolic GSH-T Activity in Hypophysectomized Male Rats

following Hormone Administration. The administration of LH,FSH, FSH plus LH, or testosterone (100 /ig 3 times daily) for 5,10, or 15 days had no significant effect on testicular cytosolicGSH-T activity determined with 4,5-BPO as substrate (Table

6).Histology. Fifty-five days after hypophysectomy of male rats,

severe abnormalities were noted in the histology of testis,relative to sham-operated control animals. Leydig cell cyto-plasmic volume was significantly decreased with concomitantappearance of dense nuclear heterochromatin in the elongatednuclei as a result of cytoplasmic loss. The interstitial space was

JULY 1980 2489



/. P. Lee et al.

Table 6Effects of the selected hormone administration on testicular GSH-7 activity in

hypophysectomized male ratsGSH-T activity was determined using [!H]benzo(a)pyrene 4.5-oxide as a

substrate. Testicular GSH-T activity was determined from the pooled testes from4 to 6 hypophysectomized male rats for each assay.

GSH-T activity (nmol conjugates/min/mg microsomal protein) at following treatment times

HormonesO.tSMNaCIFSHLHFSH

+LHTestosterone5

days18.90Â±0.87a17.67

Â±1.2517.29Â±1.251

7.93 Â±1.2921.61Â±0.411

0days24.32

Â±1.7517.83 Â±1.3623.46

Â±1.8021.92Â±2.1723.74Â±3.8315

days22.81

Â±1.701772 Â±09320.03

Â±1.5716.21Â±2.1918.38Â±1.03

a Mean Â±S.D. of 3 experiments for all values.

reduced in volume, and densely clustered heterochromaticnuclei were seen without any apparent decrease in the numberof Leydig cells (Fig. 1, A and B). In addition, the seminiferoustubule diameter was significantly decreased with loss of sper-

matogenic cells. Numerous degenerative spermatogenic cellnuclei were observed. Three daily i.p. injections of FSH (300!itg daily) for 15 days failed to alter Leydig cell volume but theheterochromatic nuclear morphology appeared to change tomore pyknotic nuclei, and in some tubules premeiotic spermatogenic cells appeared to recover in number (Fig. 1, C andD). In contrast, the dramatic morphological recovery of Leydigcells was achieved by LH treatment (3 daily injections of LH for15 days) as shown in Fig. 1, Â£and F. Similarly, FSH and LHadministered together resulted in the morphological recoveryof Leydig cells as well as a greater recovery of spermatogeniccells than caused by LH treatment alone (Fig. 1, G and H),evidenced by a significant increase in the diameter of seminiferous tubules. Although not demonstrated in Fig. 1, G or H, anincreased number of premeiotic spermatocytes and spermatidswas also found in many tubules after administration of LH plusFSH.

Testosterone treatment had no significant effect on Leydigcell morphology, although a partial recovery of spermatocyteswas apparent (Fig. 1, / and J).

DISCUSSION

We have previously reported that AHH activity and cyto-chrome P-450 content of microsomes prepared from interstitialcells of rat testis were approximately 2-fold higher than in

microsomes prepared from spermatogenic cells. Also, we reported that the EH and GSH-T activities were about 2-fold

higher in microsomal and cytosolic fractions from the spermatogenic cells than in interstitial cells (31 ). Similarly, cytochromeP-450-dependent steroid-metabolizing enzymes such as 17a-hydroxylase and Ci7-2o-lyase have been found to be predominantly associated with Leydig cell microsomes (30, 35). Sincecytochrome P-450-dependent androgen biosynthesis in Leydigcells of rat testis is under LH regulation (4), the present studywas undertaken to determine the effects of LH, FSH, andtestosterone on cytochrome P-450 and AHH activity in testis

of hypophysectomized male rats, due to our interest in thebiotransformation of pollutants by steroidogenic tissues. Wehave also investigated the effects of administering the abovehormones on microsomal EH and GSH-T activity in testes of

hypophysectomized rats, since these enzymes are important

in the further metabolism of toxic alkene and arene oxides.The results demonstrated that, following hypophysectomy,

specific testicular microsomal AHH and EH activities and cytochrome P-450 content, but not cytosolic GSH-T activities,

were significantly reduced coincident with decreased Leydigcell volume and germ cell degeneration. However, LH hormonetreatment for 15 days following hypophysectomy (40 to 55days posthypophysectomy, 110 to 125 days old) dramaticallyincreased testicular AHH and EH activities and cytochrome P-

450 content. In contrast, FSH or testosterone treatment hadno effect on testicular AHH and EH activities and cytochromeP-450 levels. Thus, it appears that increases of testicular AHHand EH activities and cytochrome P-450 content are regulated

by LH hormone and that the induction of these enzymes inhypophysectomized rats is primarily associated with markedincreases in the cellular volume of Leydig cells. On the otherhand, specific cytoplasmic GSH-T activity (at least with 4,5-

BPO as the substrate) in testis from hypophysectomized ratswas not affected by administration of LH, FSH, or testosterone.

The extent of LH-mediated recovery of AHH and EH activitiesand cytochrome P-450 content in testicular microsomes from

hypophysectomized male rats was dramatic, and it was highestin the 15-day treatment group [14- 5-, and 10-fold that of 0.15M NaCI-treated hypophysectomized rats (control), respec

tively].The LH induction of testicular microsomal AHH, EH, and

cytochrome P-450 was paralleled by morphological recovery

of the Leydig cell compartment. The recovery of cytoplasmicvolume and nuclei in hypophysectomized rats following LHtreatment was quite remarkable, as reported previously byothers (33, 37).

When FSH and LH were administered to hypophysectomizedmale rats, lower specific testicular AHH and EH activities anddecreased cytochrome P-450 contents were observed than inhypophysectomized rats treated with LH alone (Tables 3 and4). This observation apparently is due to an increase of microsomal protein in the germ cells by FSH treatment that has littleor no EH, AHH, and cytochrome P-450. The action of FSH on

male gonads has been demonstrated to stimulate Sertoli cellsto synthesize androgen-binding protein with the subsequent

stimulation of spermatogenic cell differentiation (14, 29). Asexpected, neither FSH nor testosterone treatment had anysignificant effect on Leydig cell morphology, testis microsomalAHH and EH activities, or cytochrome P-450 levels.

The molecular basis by which LH induces cytochrome P-450

levels, AHH, and EH enzymes is not clear at the present time.However, interaction between LH receptors of Leydig cells andLH hormone increases intracellular cyclic adenosine 3',5'-

monophosphate concentration with subsequent stimulation oftestosterone biosynthesis (6, 36) accompanied by increasedcytochrome P-450 content, and 17a-hydroxylase and Ci7-2o-lyase activities in the hypophysectomized rat (35). Thus, LH-associated increases of cytochrome P-450 and EH appear tobe related to the LH induction of cytochrome P-450-dependentsteroid-biosynthetic enzymes. An analogous finding has beenreported recently in which administration of adrenocortico-trophic hormone to hypophysectomized male Sprague-Dawley

rats restored adrenal microsomal AHH activity (13). Moreover,adrenal AHH was both catalytically and electrophoreticallysimilar to hepatic 3-methylcholanthrene-induced AHH but isdistinctively different from that of progesterone C2,-hydroxyl-





ase. In contrast to Sprague-Dawley rat testes, however, AHH

in adrenal microsomes is not induced, either in the intact or inthe hypophysectomized animals, by 3-methylcholanthrene orby 2,3,7,8-tetrachlorodibenzo-p-dioxin. In rat testes, however,

in addition to LH induction of monooxygenase activity, otherenvironmental chemicals such as PAH and 2,3,7,8-tetrachlo-rodibenzo-p-dioxin have recently been shown to increase tes-ticular AHH activity from 1.5- to 2-fold in Sprague-Dawley rats(21) and from 85- to 500-fold in C57BL/6 mice (27), respectively. Thus, PAH inducibility of microsomal AHH in varioustarget tissue of hormones is demonstrated to be different.Since interstitial cell tumors of the testis have been suggestedto be due to the differential presence of steroidogenic enzymesassociated with mixed-function oxidase system(s) in Leydig

cells of both rodents (8, 15) and humans (5, 23), it is temptingto speculate that environmental factors affecting differentialactivities of testicular mixed-function oxidases system(s) intestes may play an important role in the chemical carcinogen-

esis and genetic toxicity of male gonads.

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JULY 1980 2491



/. P. Lee et al.

B

*IS Â«Â«v-- ;

V

Fig. 1. Testicular histology of hypophysectomized control and the following various hormone treatments. Arrows. Leydig cells. A. 55 days after hypophysectomy.Leydig cells and degenerative spermatocytes. x 500. ÃŸ,loss of cytoplasmic volume along with heterochromatic nuclei, x 1125. C and D, FSH treatments, somemorphological recovery of spermatocytes with further changes in Leydig cell nuclear change. C. x 500; D. x 1,125. Â£and F, LH treatment dramatic morphologicalrecovery of both spermatogenic cells and Leydig cells with respect to cytoplasmic volume and nuclear structure. E. x 500; F, x 1125. G and H. FSH plus LHtreatment, some recovery of spermatogenic cells with respect to both morphology and cell numbers. Leydig cell morphology is similar to that of LH treatment alone.G, x 500; and H. x 1125. / and J, testosterone treatment, some morphological recovery of spermatocytes without any morphological changes of Leydig cells. /, x500: J. x 1125.




1980;40:2486-2492. Cancer Res Insu P. Lee, Katsushi Suzuki, Hasan Mukhtar, et al. Activities in Testis of Hypophysectomized RatsMonooxygenase Activity and Epoxide-metabolizing Enzyme Hormonal Regulation of Cytochrome P-450-dependent

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