Effects of dimethylnitrosamine on some actions of testosterone

Toxico/ogy

Letters,

133

8 (1981) 133-139

Elsevier/North-Holland

Biomedical

Press

EFFECTS OF DIMETHYLNITROSAMINE TESTOSTERONE

F.I. IKEGWUONU, Department

November

(Revision

received

(Accepted

January

OF

T.A. AIRE and S.O. OGWUEGBU

of Veterinary

(Received

ON SOME ACTIONS

Anatomy,

University

of Ibadan,

Ibadan

(Nigeria)

3rd, 1980) January

8th, 1981)

11 th, 198 1)

SUMMARY Using homogenates of mouse kidney and testes, the activities of the enzymes, galactosidase, were studied as markers of androgen action. The results obtained and

kidney

anabolic

homogenates.

Dimethylnitrosamine

action of testosterone

(DMN)

in kidney homogenates

may cause

fl-glucuronidase and pdiffered between testes

a competetitive

inhibition

of the

but this was not evident from the results obtained

with testes homogenates.

INTRODUCTION

The occurrence

of nitrosamines

in foodstuffs

has been documented

extensively

[l-3]. Under laboratory conditions, they have been found to be both mutagenic and carcinogenic [4, 51. A feature of the carcinogenicity of nitrosamines in animals is an ability to induce various types of tumours in the progeny of treated parents [6-81. The data obtained from animals experiments suggest that nitrosamines may have some effects on androgen action. Testosterone administration induces a marked, consistent and specific anabolic response through an increase in RNA and protein synthesis in the mouse kidney [9]. The use of mouse kidney homogenates is now an accepted ex vivo system for the study of androgen action. The purpose of this investigation was to attempt to study the effects of DMN on the activities of the enzyme markers of androgen action, pglucuronidase and fl-galactosidase [IO]. To correlate enzyme activity with protein synthesis, the concentration of kidney total protein, RNA and DNA were

Abbreviations:

ANOVA,

analysis

037884274/81/0000~0000/$02.50

of variance;

DMN, dimethylnitrosamine.

0 Elsevier/North-Holland

Biomedical

Press

134

determined.

Similar

assess differences MATERIALS

AND

experiments

were performed

in organ/tissue

responsiveness.

with mouse testes homogenates

to

METHODS

36 adult male mice of the albino strain weighing between 26-35 g and inbred in the animal house of the Faculty of Veterinary Medicine (University of Ibadan, Ibadan) were allocated randomly to 3 groups, at the rate of 12 mice per group. Group A mice received, every 3 days, an S.C. injection of 0.1 ml (16 mg/kg body weight) testosterone dissolved in groundnut oil. Group B mice received the same dose but with an additional i.p. injection of 0.02 ml (20 +g) of DMN on day 4. Group C animals served as controls and each mouse received 0.1 ml groundnut oil only on every experimental day. Each animal was weighed after each treatment. Nine treatments were given to each mouse. All the animals received food and water ad lib throughout the experimental period.

Tissue homogenates At the end of the treatments,

each animal

was stunned

and decapitated.

The

kidneys and testes were removed, blotted dry and weighed. The organs were quickly dropped in an ice-cold (0-4°C) solution of 0.25 M sucrose. The testes were homogenised as previously described [I l] and the homogenisation of the kidneys was done according to the method of Bardin et al. [ 121. Homogenate concentration used for analyses was 20 mg/ml.

Tissue analyses 2 ml of each of the homogenates, made alkaline with an equal volume of 0.67 N NaOH, were used to analyse tissue RNA, DNA and total protein. 3.8 ml of this new solution were used for RNA and DNA estimation, while 0.2 ml was utilised for total protein measurement. RNA was determined after perchloric acid hydrolysis using the method of Munro and Fleck [13]. DNA was estimated in the supernatant from RNA determination by the method of Burton [14]. Highly polymerised calf thymus DNA was used as a standard. Total protein was measured in the tissues by the procedure of Lowry et al. [ 151, using bovine serum albumin (fraction V) as the standard protein. Calculations of these parameters were extrapolated in each case to g of wet tissue.

Enzyme determinations &Glucuronidase and @-galactosidase activities were assayed by a modified method of Meisler and Paigen [ 161. The volumes of 30% trichloroacetic acid and 2-

135

amino-2-methyl-1,3-propanediol were increased and added to the incubation mixture which consisted of 0.25 ml of a solution of 0.56% Triton X-100 + 0.56 M NaOAc; 0.1 ml of 10 mM substrate; 0.6 ml TCA; 0.6 ml 2-amino-2-methyl-1,3propanedioi. The substrates used were p-nitrophenyl-P-D-glucuronide and pnitrophenyl-/3-D-galactopyranoside, respectively. One unit of enzyme activity was defined as that amount releasing 1 nm of p-nitrophenyl/h at 56°C (for mice) and expressed as units/g wet tissue. Statistical analysis A two-factor ANOVA as described by Yamane [17] was used to analyse the data. RESULTS Apart from transient weight loss in mice receiving testosterone + DMN associated with the second treatment (day 6), there were no conclusive effects on body weight (Fig. 1). It can be seen from Fig. 2 that while the administration of testosterone alone decreased the weight of the testis (P = 0.02) testosterone + DMN caused an increase in the weight of this organ (P = 0.08). These changes were not statistically significant (P = 0.05). On the contrary, the testosterone- induced increases in

Controlrn,ce ~ --

I

2(6)

I

Fig.

1. Bodyweight

mean body weight

I

I

4(12) 6(18) NUMBER OF TREATMENTS

I

8(24)

curves of mice given testosterone

of 12 mice. The numbers

Mice treated with testosterone only Mace treated with testosterone and dimethyl nltrosamlne (DMN)

lO(30)

or testosterone

in parentheses

+ DMN. Each point represents

on the abscissa

represent

treatment

days.

the

kidney weight (P = 2.1, significant), were apparently enhanced by DMN (P = 2.2, significant). Testosterone administration induced a 20-fold and approx. 15fold, increase in kidney homogenate ~-g~u~uronidase and @-galactosidase activity respectively (Table I). Homogenates from animals given testosterone and DMN showed only a 5-fold increase over the control data. Similar patterns emerged for the measurement of total protein and RNA. However, DMN did not diminish the effect of testosterone on DNA content of the homogenate. The responses evident in testis homogenates differed from the results obtained with kidney homogenates (Table II). DMN augmented rather than inhibited the enzyme activity detectable in testis homogenates with testosterone. However, increased concentration of the rise in testis homogenate of mice given testosterone were less obvious in mice given DMN. DISCUSSION

The differing responses of kidney and testicular homogenates are not entirely surprising since the physiological role of testosterone is quite different for these increases in the enzyme parameters organs. The initial testosterone-induced investigated were consistent with the expected activity of administering exogenous

Control m,ce b$g; gbven testosterone Mice treated with k?StoStefone and dlmethy! nlttosamlne CDMN)

~:ii

~

r,~ F=i

TESTIS.WEIGHTS

Fig. 2. Differences DMN.

Control

significantly the control

KIONEY WEIGHTS

in the testis and kidneys weights of mice injected with testosterone

values are included

differ

for comparison

from the controi (P > 0.05).

(P i 0.001).

purposes.

Bars marked

or testosterone

with one asterisk

Bars marked with two asterisks significantly

differ

+

do not from

83.8 k 2.2a.c 50.4 * 1.5

the mean of 12 determinations f SE. from control (P < 0.01) from control (P < 0.05) from Group A treatment (P 4 0.05)

298.4 k 8.2b 247.3 + 4.3

60.7 it 1.8b

Group A (testosterone) Group B (testosterone) + DMN Group C (controls)

Each value represents asignificantly different %ignificantly different CSignificantly different

280.2 + 6.4b

/.?-Glucuronidase (units/g)

Treatments

80.1 k l.Sa+’ 66.4 f 2.1

98.6 k 4.7a

(m&g)

Total protein

2.0 + 0.Ib.d 1.2 rf 0.1

3.4 t 0.3b

RNA (mg/g)

4.4 + 0. lb 2.7 k 0.1

4.5 k 0.3b

DNA (m&g)

36.3 z!z1.8b,’ 30.6 k 1.7

42.4 + 2.2a

Total Protein (mg/g)

1.3 i O.lb 1.1 rt 0.1

1.9 t-O.lb

RNA (m&g)

1.6 f 0.3 1.6 + 0.3

2.2 f 0.4”

DNA (mg/g)

(DMN) ON SOME INDICATORS OF ANDROGEN ACTION IN MOUSE TESTIS

p-Galactosidase (units/g)

EFFECTS OF TESTOSTERONE AND DIMETHYLNITROSAMINE HOMOGENATES

TABLE II

the mean of 12 determinations t S.E. from control (P < 0.01) from control (P < 0.05) from Group A treatment (P < 0.01) from Group A treatment (P < 0.05)

302.6 r?:4.la$c 60.6 z%2.3

192.5 rt 1.8a,c 38.9 + 1.1

Each value represents asignificantly different bignificantly different CSignificantIy different Qignificantly different

910.7 4 8.3a

780.2 F 3.@

Group A (testosterone) Group B (testosterone) + DMN Grouo C (controls)

@Galactosidase (units/g)

~-Glucuronidase (units/g)

(DMN) ON SOME INDICATORS OF ANDROGEN ACTION IN MOUSE KIDNEY

Treatments

EFFECTS OF TESTOSTERONE AND DIMETHYLNITROSAMINE HOMOGENATES

TABLE I

5

138

testosterone.

The differing

activity of testosterone testosterone may affect Our results of kidney

indicate

responses

to DMN not only reflect the primary

in kidney tissue but also demonstrate the testes paradoxically. that DMN has an inhibitory

homogenates

to testosterone.

anabolic

that high doses 01

effect on the anabolic

The diminished

response

RNA and protein

responses

following additional DMN administraton may be explicable on the basis that some of the testosterone induced RNA may have been alkylated by DMN [18]. Since DMN did not affect kidney homogenate response to testosterone, the cause for the increase in kidney weight (Fig. 2) is not readily apparent. P-Glucuronidase activity correlated negatively with spermatogenesis [ 191 as may be /3-galactosidase activity since both enzymes act in the same pathways [16]. The data show that testosterone might increase activity of these enzymes in testes homogenates which may be diminished by DMN. This phenomenon is not easily explained and is worthy of further study. Since androgen-induced changes in mouse kidney homogenates are mediated by functional androgen receptors [20], these results may suggest an effect of DMN on androgen receptor binding, i.e. a form of competitive inhibition. These preliminary results indicate a need for further work to explain the mechanism of the interaction between DMN and androgen-dependent processes and other effects seen in animal experiments. ACKNOWLEDGEMENTS

The authors are grateful to Dr. E.N. Maduagwu of the Biochemistry Department, University of Ibadan, Ibadan for the gift of dimethylnitrosamine used for this work.

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