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Effects of intratesticular aflatoxin B1 on rat testes and blood estrogens
263
Twxicofogy Letters, 5 (1980) 263-267 Q Elsevier/North-Holland Biomedical Press
EFFECTS OF INTRATESTICULAR AND BLOOD ESTROGENS
AFLATOXIN B, ON RAT TESTES
T. GOPAL, F.W. OEHME, T.F. LIAO and C.L. CHEN C~m~arat~ve Toxicot~gy ~ab#ra~~ries, College University, ~anhaftan, KS 66506 (U.S.A.)
of Veterinary
~e~~e~ne,
Kansas State
(Received September Z&h, 1979) (Accepted November 8th, 1979)
-_.SUMMARY
An investigation of the effects of aflatoxin B1 on rat testes and plasma estrogens is reported. A normal saline-suspended aflatoxin B, was injected into right testes of mature Sprague-Dawley rats at doses of 0, 5, 10, 25 or 50 pg. Effects were evaluated by histopathologi~~~terations of the testes and by levels of plasma estrogens estimated by a modified Dextron-Ch~~oal radioimmune assay. Significant gross lesions were present in testes injected with the 50 pg dose. There was a 50% reduction in size and weight as compared to the left testis. Histological changes were dose-dependent, with minimal or no changes at 5 pg and maximal effects at 50 pg. The lesions varied from mild testicular degeneration to complete disappearance of cellular components. Interstitial cell proliferation, occasional calcification of seminiferous tubules, and absence of spermatogenesis and spermatozoa were extreme toxic effects. Estrogen concentrations in injected rats were reduced compared to those of control and normal rats. This study suggested that following intratesticular aflatoxin B1 exerts a direct toxic effect on testes with a reduction in plasma estrogen levels.
INTRODUCTION
Aflatoxins, a group of fungal metabolites, have been incriminated as primary hepatotoxins and as causing acute and chronic toxic effects in most animal species and man [l] . They also induce genetic effects by affecting the DNA synthesis [2]. Genetic damage from aflatoxins can also result in carcinogenic, teratogenic and mutagenic effects (3-71. Sex hormones have been found to exert profound potentialities in modifying the toxicity and carcinogenicity of aflatoxins [S, 91. Reduced toxic effects of aflatoxin in castrated male rats have also been reported [lo]. Increased hepatie injury was noticed when aflatoxin was administered together with
264
androgens. Testicular atrophy and azoospermia have been reported in male rats fed on aflatox~-contaminated feed [ll] . More recently intraperitoneal injections of aflatoxin B1 in male rats failed to produce any recognizable adverse effects on the reproductive system [12]. It is not understood whether aflatoxins exert their toxic effects directly on germinal epithelium or indirectly via hepatic injury resulting in testicular atrophy. Several studies in humans have shown testicular atrophy with elevated plasma estrogen levels [13]. Hence the present investigation studied the effect of intratesticular inoculation of aflatoxin B1 on the testes of mature male rats. The effects were evaluated by histological examinations and biochemical determination of plasma estrogens. MATERIALS
AND METHODS
of the toxin for injection A uniform suspension of aflatoxin B1 was prepared in phosphate buffer (pH 7.2) to contain 50 pg. Treatment of crystalline aflatoxin with chloroform and methanol before adding it to the buffer produced a uniform suspension. Chloroform was evaporated by placing the suspension in a water bath at 60°C for 5 to 10 min. The concentration of aflatoxin in the suspension was determined by TLC using standard solution of aflatoxin B1.
Preparation
Treatment in ruts Mature, male Sprague-Hawley rats were used. The aflatoxin suspension was injected into the right testis of each rat at dosage levels of 5,10,25, or 50 E.tgrespectively; controls were given injections of buffer, buffer and methanol, and buffer, methanol and chloroform (Table I). TABLE I EFFECTS OF TESTICULAR
~__________
INJECTIONS OF AFLATOXIN
Treatment .____~ Needle puncture Buffer (Buf) Buf. and methanol (meth) Buf. meth. and chloroform (chl) Buf. meth. chl. and aflatoxin Buf. meth. chl. and aflatoxin Buf. meth. chl. and aflatoxin Buf. meth. chl. and aflatoxin Buf. meth. chl. and aflatoxin __I-... .-_
B, IN RATS
Volume (ml) __._
Aflatoxin kg)
Lesions
0.0 0.2 0.2 0.2 0.1 0.2 0.2 0.5 1.0
0 0 0 0 5 10 10 25 50
none none none none v. slight slight slight moderate complete
265
~eterm~nation o~~l~rna estrogens 3 ml of whole blood was collected by intracardiac puncture from normal rats for pre-treatment estrogen assay. Blood sampling was done on the experimental rats on the 11th day before sacrifice. Samples were assayed for plasma total estrogen by a modified dextron-charcoal radioimmune assay (RIA) technique and the results expressed in pg/ml 114,151. Gross and histological studies Rats were euthanized on the 11th day. Testes were examined, individually weighed, and gross changes recorded. They were then fixed in 10% buffered formalin for histological study. RESULTS
Significant gross changes were observed in the testes given the 50 Rg dose. They appeared shrunken and partly adhered to the abdominal cavity. There was a 50% reduction in weight as compared to the contralateral left testis. The weight of the testis was slightly reduced at the 25 and 10 pg doses, but no gross changes were seen. No change was noticed at the 5 pg dose. The testes of control rats did not show any significant gross change. Histologically, the changes were maximal at the 50 pg dose level (Fig. 1). A marked absence of cellular components were made in the seminiferous
Fig. 1. Photomicrograph of the right testes of a rat that was injected with aflatoxin B, 11 days previously. H & E, 100 x .
50
mg of
266
tubules with infiltration of homogenous hyalinized eosinophilic material. Interstitial cells showed slight proliferative changes. Calcification of many seminiferous tubules was evident. Spermatozoa were completely absent in the lumen of most of the tubules at this dose level. The changes varied from moderate to slight as the dose levels decreased from 25 pg to 5 p. Marked degenerative changes were noticed at 25 pg although they were at a lesser degree than those at 50 pg. Few tubules had undergone complete degeneration, and the remaining tubules appeared normal with evidence of spermatogel~esis. Normal spermatozoa were seen in the unaffected seminiferous tubules. No significant histological alterations were seen in the left testes of the aflatoxin-treated rats and in bath testes of control rats. Healed lesions with mild lymphocytic reaction were noticeable at the site of inoculation in the right testes of all injected rats (Table I). The plasma estrogen concentration of aflatoxin-injected rats was reduced by 18% compared to those of control and normal rats. The levels in the control rats remained the same as those of normal rats (Table II). TABLE II PLASMA ESTROGEN LEVELS (pg/mi) IN NORMAL, CONTROL AND AFLATOXININJECTED RATS .__ . .._-._ _ ~._~~_..~ .-..- ..._.-. .--.-Normal
Control
14.0 9.6 11.8 14.0 16.7 13.2 ....
16.5 10.5 14.0 11.9
_--~--_
Mean SD SEM .... ..--- . ..-_ “_..”
Aflatoxin-injected
.____ ..__.. ~.
--
13.2 2.3
1.1
._~..~ ..-.-. _--”
..____.__._-__.-
13.0 8.8 11.5 9.3 11.6 1O.P 1.6 0.7
al&2% reduction in estrogen Iesef (Y’
_--_...
~._.._~.
-. _____..___. --
= 1.820; 0.1 > P >0.05).
DPSCUSSION
Microscopic changes were significant at aflatoxin dose levels of 25 and 50 ,ug. The minimal histological changes observed at a 10 pg dose level and below might prove of greater significance if genetic damage were produced. Although the number of animals used in this study is too small to permit any definite conclusions, it may be presumed that molecular changes could occur resulting in genetic damage without affecting reproductive efficiency. Although the maximum effects of aflatoxins are due to their primary hepatotoxic activity, lower levels of aflatoxin could affect other systems, and particularly the reproductive system, with disastrous consequences.
267
The reduced levels of plasma estrogens suggest the effect of aflatoxin on the testes is a sequel to direct parenchymatous damage and not indirectly due to hepatic disorder. In chronic dietary hepatic disease in humans, atrophy of the testes and gynecomastia result from increased levels of circulating estrogens [13, 161. Either or both of these factors are associated with altered metabolic and enzymic activity from diseased hepatic parenchyma. The testicular atrophy in the present study resulted from direct action of the injected aflatoxin. The reduced levels of estrogens suggest that metabolic activity of the liver was normal. Injury to testicular parenchyma may also have resulted in reduced synthesis of testicular estrogens [17]. REFERENCES 1 2 3
L.A. Goldblatt, Aflatoxins, Academic Press, New York, 1969. M.S. Legator, Biological assay for aflatoxins, in L.A. Goldblatt (Ed.), kflatoxins, Academic Press, New York, 1969, pp. 107-147. J.M. Barnes and W.H. Butler, Carcinogenetic activity of aflatoxin in rats, Nature, (1964)
4 5 6 7 8 9 10
11 12 13
14
15
202
1016.
F. Dickens and H.E.H. Jones, The carcinogenic action of aflatoxin after its subcutaneous injection in the rat, Br. J. Cancer, 17 (1964) 691-698. J.A. DiPaolo, J. Ellis and H. Enwin, Teratogenic response by hamsters, rats and mice to aflatoxin B,, Nature, 215 (1967) 638-639. J. Elis and J.A. DiPaolo, Aflatoxin B, : induction of malformations, Arch. Pathol., 83 (1967) 53-57. M.S. Legator, S.M. Zuffante and A.R. Harp, Aflatoxin effect in cultured heteroploid human embryonic lung cells, Nature, 208 (1965) 345-348. G.N. Wogan and P.M. Newberne, Dose response characteristics of aflatoxin B, in the rat, Cancer Res., 27 (1967) 2370-2376. P.M. Newberne and G. Williams, Inhibition of aflatoxin carcinogenesis by diethyl stilbesterol in male rats, Arch. Environ. Health, 19 (1969) 489-498. H.F. Righter, W.T. Shalkop, D.H. Mercer and E.C. Leffel, Influence of age and sexual status on the development of toxic effects in male rats fed aflatoxins, Toxicol. Appl. Pharmacol., 21 (1972) 435-439. C. Richer, M. Matinend, T. Toury and H. Dupin, Sur les effets canc&rig&es de rCgimes contenant des arachides contamindes, C. R. Sot. Biol. 158 (1965) 1375-1378. G.N. Egbunike, The effects of micro doses of aflatoxin B, on sperm production rates, epididymal sperm abnormality and fertility in the rat, Zbl. Vet. Med., 26 (1979) 66-72. A. Galvao-Teles, D.C. Anderson, C.W. Burke, J.C. Marshall, C.S. Corker, R.L. Bown and M.L. Clark, Biologically active androgens and oestradiol in men with chronic liver disease, Lancet, 1 (1973) 173-177. A.R. Midgley Jr. and G.D. Niswender, Radioimmune assay of steroids, in E. Diczfalusy (Ed.), Karolinski Symposia on Methods in Reproductive Endocrinology, 2nd Symposium: Steroid Assay by Protein Binding, 1970, pp. 32-355. G. Mikhail et al., Radio immune assay of estrogens and estradiol, Acta Endocrinol., 64 Suppl. 1970, 147-347. Cited by Larson, S.M. and Thorell, J.T., Principles of competitive radio assay, Lab. Med., 5 (1974) 15-21.
16
J.R. Brown, G.P. Crean and J. Ginsburg, Oestrogen metabolism and excretion in liver disease, Gut, 5 (1964) 56-59.
17
W.R. Gomes, Metabolic and regulatory hormones A.D., Gomes, W.R. and Vandemark, N.L. (Eds.), New York, 1970.
influencing Testes Vol.
testes function, in Johnson, III, Academic Press,
Twxicofogy Letters, 5 (1980) 263-267 Q Elsevier/North-Holland Biomedical Press
EFFECTS OF INTRATESTICULAR AND BLOOD ESTROGENS
AFLATOXIN B, ON RAT TESTES
T. GOPAL, F.W. OEHME, T.F. LIAO and C.L. CHEN C~m~arat~ve Toxicot~gy ~ab#ra~~ries, College University, ~anhaftan, KS 66506 (U.S.A.)
of Veterinary
~e~~e~ne,
Kansas State
(Received September Z&h, 1979) (Accepted November 8th, 1979)
-_.SUMMARY
An investigation of the effects of aflatoxin B1 on rat testes and plasma estrogens is reported. A normal saline-suspended aflatoxin B, was injected into right testes of mature Sprague-Dawley rats at doses of 0, 5, 10, 25 or 50 pg. Effects were evaluated by histopathologi~~~terations of the testes and by levels of plasma estrogens estimated by a modified Dextron-Ch~~oal radioimmune assay. Significant gross lesions were present in testes injected with the 50 pg dose. There was a 50% reduction in size and weight as compared to the left testis. Histological changes were dose-dependent, with minimal or no changes at 5 pg and maximal effects at 50 pg. The lesions varied from mild testicular degeneration to complete disappearance of cellular components. Interstitial cell proliferation, occasional calcification of seminiferous tubules, and absence of spermatogenesis and spermatozoa were extreme toxic effects. Estrogen concentrations in injected rats were reduced compared to those of control and normal rats. This study suggested that following intratesticular aflatoxin B1 exerts a direct toxic effect on testes with a reduction in plasma estrogen levels.
INTRODUCTION
Aflatoxins, a group of fungal metabolites, have been incriminated as primary hepatotoxins and as causing acute and chronic toxic effects in most animal species and man [l] . They also induce genetic effects by affecting the DNA synthesis [2]. Genetic damage from aflatoxins can also result in carcinogenic, teratogenic and mutagenic effects (3-71. Sex hormones have been found to exert profound potentialities in modifying the toxicity and carcinogenicity of aflatoxins [S, 91. Reduced toxic effects of aflatoxin in castrated male rats have also been reported [lo]. Increased hepatie injury was noticed when aflatoxin was administered together with
264
androgens. Testicular atrophy and azoospermia have been reported in male rats fed on aflatox~-contaminated feed [ll] . More recently intraperitoneal injections of aflatoxin B1 in male rats failed to produce any recognizable adverse effects on the reproductive system [12]. It is not understood whether aflatoxins exert their toxic effects directly on germinal epithelium or indirectly via hepatic injury resulting in testicular atrophy. Several studies in humans have shown testicular atrophy with elevated plasma estrogen levels [13]. Hence the present investigation studied the effect of intratesticular inoculation of aflatoxin B1 on the testes of mature male rats. The effects were evaluated by histological examinations and biochemical determination of plasma estrogens. MATERIALS
AND METHODS
of the toxin for injection A uniform suspension of aflatoxin B1 was prepared in phosphate buffer (pH 7.2) to contain 50 pg. Treatment of crystalline aflatoxin with chloroform and methanol before adding it to the buffer produced a uniform suspension. Chloroform was evaporated by placing the suspension in a water bath at 60°C for 5 to 10 min. The concentration of aflatoxin in the suspension was determined by TLC using standard solution of aflatoxin B1.
Preparation
Treatment in ruts Mature, male Sprague-Hawley rats were used. The aflatoxin suspension was injected into the right testis of each rat at dosage levels of 5,10,25, or 50 E.tgrespectively; controls were given injections of buffer, buffer and methanol, and buffer, methanol and chloroform (Table I). TABLE I EFFECTS OF TESTICULAR
~__________
INJECTIONS OF AFLATOXIN
Treatment .____~ Needle puncture Buffer (Buf) Buf. and methanol (meth) Buf. meth. and chloroform (chl) Buf. meth. chl. and aflatoxin Buf. meth. chl. and aflatoxin Buf. meth. chl. and aflatoxin Buf. meth. chl. and aflatoxin Buf. meth. chl. and aflatoxin __I-... .-_
B, IN RATS
Volume (ml) __._
Aflatoxin kg)
Lesions
0.0 0.2 0.2 0.2 0.1 0.2 0.2 0.5 1.0
0 0 0 0 5 10 10 25 50
none none none none v. slight slight slight moderate complete
265
~eterm~nation o~~l~rna estrogens 3 ml of whole blood was collected by intracardiac puncture from normal rats for pre-treatment estrogen assay. Blood sampling was done on the experimental rats on the 11th day before sacrifice. Samples were assayed for plasma total estrogen by a modified dextron-charcoal radioimmune assay (RIA) technique and the results expressed in pg/ml 114,151. Gross and histological studies Rats were euthanized on the 11th day. Testes were examined, individually weighed, and gross changes recorded. They were then fixed in 10% buffered formalin for histological study. RESULTS
Significant gross changes were observed in the testes given the 50 Rg dose. They appeared shrunken and partly adhered to the abdominal cavity. There was a 50% reduction in weight as compared to the contralateral left testis. The weight of the testis was slightly reduced at the 25 and 10 pg doses, but no gross changes were seen. No change was noticed at the 5 pg dose. The testes of control rats did not show any significant gross change. Histologically, the changes were maximal at the 50 pg dose level (Fig. 1). A marked absence of cellular components were made in the seminiferous
Fig. 1. Photomicrograph of the right testes of a rat that was injected with aflatoxin B, 11 days previously. H & E, 100 x .
50
mg of
266
tubules with infiltration of homogenous hyalinized eosinophilic material. Interstitial cells showed slight proliferative changes. Calcification of many seminiferous tubules was evident. Spermatozoa were completely absent in the lumen of most of the tubules at this dose level. The changes varied from moderate to slight as the dose levels decreased from 25 pg to 5 p. Marked degenerative changes were noticed at 25 pg although they were at a lesser degree than those at 50 pg. Few tubules had undergone complete degeneration, and the remaining tubules appeared normal with evidence of spermatogel~esis. Normal spermatozoa were seen in the unaffected seminiferous tubules. No significant histological alterations were seen in the left testes of the aflatoxin-treated rats and in bath testes of control rats. Healed lesions with mild lymphocytic reaction were noticeable at the site of inoculation in the right testes of all injected rats (Table I). The plasma estrogen concentration of aflatoxin-injected rats was reduced by 18% compared to those of control and normal rats. The levels in the control rats remained the same as those of normal rats (Table II). TABLE II PLASMA ESTROGEN LEVELS (pg/mi) IN NORMAL, CONTROL AND AFLATOXININJECTED RATS .__ . .._-._ _ ~._~~_..~ .-..- ..._.-. .--.-Normal
Control
14.0 9.6 11.8 14.0 16.7 13.2 ....
16.5 10.5 14.0 11.9
_--~--_
Mean SD SEM .... ..--- . ..-_ “_..”
Aflatoxin-injected
.____ ..__.. ~.
--
13.2 2.3
1.1
._~..~ ..-.-. _--”
..____.__._-__.-
13.0 8.8 11.5 9.3 11.6 1O.P 1.6 0.7
al&2% reduction in estrogen Iesef (Y’
_--_...
~._.._~.
-. _____..___. --
= 1.820; 0.1 > P >0.05).
DPSCUSSION
Microscopic changes were significant at aflatoxin dose levels of 25 and 50 ,ug. The minimal histological changes observed at a 10 pg dose level and below might prove of greater significance if genetic damage were produced. Although the number of animals used in this study is too small to permit any definite conclusions, it may be presumed that molecular changes could occur resulting in genetic damage without affecting reproductive efficiency. Although the maximum effects of aflatoxins are due to their primary hepatotoxic activity, lower levels of aflatoxin could affect other systems, and particularly the reproductive system, with disastrous consequences.
267
The reduced levels of plasma estrogens suggest the effect of aflatoxin on the testes is a sequel to direct parenchymatous damage and not indirectly due to hepatic disorder. In chronic dietary hepatic disease in humans, atrophy of the testes and gynecomastia result from increased levels of circulating estrogens [13, 161. Either or both of these factors are associated with altered metabolic and enzymic activity from diseased hepatic parenchyma. The testicular atrophy in the present study resulted from direct action of the injected aflatoxin. The reduced levels of estrogens suggest that metabolic activity of the liver was normal. Injury to testicular parenchyma may also have resulted in reduced synthesis of testicular estrogens [17]. REFERENCES 1 2 3
L.A. Goldblatt, Aflatoxins, Academic Press, New York, 1969. M.S. Legator, Biological assay for aflatoxins, in L.A. Goldblatt (Ed.), kflatoxins, Academic Press, New York, 1969, pp. 107-147. J.M. Barnes and W.H. Butler, Carcinogenetic activity of aflatoxin in rats, Nature, (1964)
4 5 6 7 8 9 10
11 12 13
14
15
202
1016.
F. Dickens and H.E.H. Jones, The carcinogenic action of aflatoxin after its subcutaneous injection in the rat, Br. J. Cancer, 17 (1964) 691-698. J.A. DiPaolo, J. Ellis and H. Enwin, Teratogenic response by hamsters, rats and mice to aflatoxin B,, Nature, 215 (1967) 638-639. J. Elis and J.A. DiPaolo, Aflatoxin B, : induction of malformations, Arch. Pathol., 83 (1967) 53-57. M.S. Legator, S.M. Zuffante and A.R. Harp, Aflatoxin effect in cultured heteroploid human embryonic lung cells, Nature, 208 (1965) 345-348. G.N. Wogan and P.M. Newberne, Dose response characteristics of aflatoxin B, in the rat, Cancer Res., 27 (1967) 2370-2376. P.M. Newberne and G. Williams, Inhibition of aflatoxin carcinogenesis by diethyl stilbesterol in male rats, Arch. Environ. Health, 19 (1969) 489-498. H.F. Righter, W.T. Shalkop, D.H. Mercer and E.C. Leffel, Influence of age and sexual status on the development of toxic effects in male rats fed aflatoxins, Toxicol. Appl. Pharmacol., 21 (1972) 435-439. C. Richer, M. Matinend, T. Toury and H. Dupin, Sur les effets canc&rig&es de rCgimes contenant des arachides contamindes, C. R. Sot. Biol. 158 (1965) 1375-1378. G.N. Egbunike, The effects of micro doses of aflatoxin B, on sperm production rates, epididymal sperm abnormality and fertility in the rat, Zbl. Vet. Med., 26 (1979) 66-72. A. Galvao-Teles, D.C. Anderson, C.W. Burke, J.C. Marshall, C.S. Corker, R.L. Bown and M.L. Clark, Biologically active androgens and oestradiol in men with chronic liver disease, Lancet, 1 (1973) 173-177. A.R. Midgley Jr. and G.D. Niswender, Radioimmune assay of steroids, in E. Diczfalusy (Ed.), Karolinski Symposia on Methods in Reproductive Endocrinology, 2nd Symposium: Steroid Assay by Protein Binding, 1970, pp. 32-355. G. Mikhail et al., Radio immune assay of estrogens and estradiol, Acta Endocrinol., 64 Suppl. 1970, 147-347. Cited by Larson, S.M. and Thorell, J.T., Principles of competitive radio assay, Lab. Med., 5 (1974) 15-21.
16
J.R. Brown, G.P. Crean and J. Ginsburg, Oestrogen metabolism and excretion in liver disease, Gut, 5 (1964) 56-59.
17
W.R. Gomes, Metabolic and regulatory hormones A.D., Gomes, W.R. and Vandemark, N.L. (Eds.), New York, 1970.
influencing Testes Vol.
testes function, in Johnson, III, Academic Press,