Effects of the benomyl metabolite, carbendazim, on the hypothalamic-pituitary reproductive axis in the male rat

Effects of the benomyl metabolite, carbendazim, on the hypothalamic-pituitary reproductive axis in the male rat

Toxicology, 57 (1989) 173--182 Elsevier Scientific Publishers Ireland Ltd. E F F E C T S OF T H E B E N O M Y L M E T A B O L I T E , C A R B E N D A...

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Toxicology, 57 (1989) 173--182 Elsevier Scientific Publishers Ireland Ltd.

E F F E C T S OF T H E B E N O M Y L M E T A B O L I T E , C A R B E N D A Z I M , ON THE HYPOTHALAMIC-PITUITARY R E P R O D U C T I V E A X I S IN THE MALE RAT

JEROME M. GOLDMAN a, GEORGIA L. REHNBERG b, RALPH L. COOPER b, L. EARL GRAY, JR. b, JOY F. HEIN b and W. KEITH McELROY b

"NSI Technology Services, Environmental Sciences, Research Triangle Park, NC and bEndocrinology/Gerontology Section~ Reproductive Toxicology Branch, Health Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, NC (U.S.A.) (Received October 17th, 1988) (Accepted December 19th, 1988)

SUMMARY

Carbendazim (MBC), the bioactive metabolite of the fungicide benomyl, has been reported to induce a number of testicular alterations in male rats. Since it is possible that extragonadal changes contribute to the appearance of such effects, the present study focused on the presence of concurrent endocrine changes in the hypothalamic and pituitary components of the brain-pituitary-testicular axis. Subchronic administration of MBC (50, 100, 200 or 400 mg/kg) was found to cause a dose-related elevation in serum follicle stimulating hormone (FSH) and pituitary luteinizing hormone (LH). Values for prolactin and thyroid-stimulating hormone remained unchanged. No statistical differences in gonadotropin-releasing hormone concentrations were present in mediobasal hypothalamus, although an elevation in anterior hypothalamic values was found at the low dose, followed by a dose-related decline. These findings demonstrate that previously reported gonadal differences following subchronic exposure to carbendazim are accompanied by alterations elsewhere in the reproductive system which appear to involve both changes in Sertoli cell-pituitary feedback signals and direct effects of the compound on the central nervous system.

Key words: Methyl 2-benzimidazole carbamate; Carbendazim; Fungicide; Reproductive system; Pituitary; Hypothalamus; Hormones Address all correspondence and reprint requests to: Jerome M. Goldman, Ph.D., MD-8, NSI Technology Services, Environmental Sciences, Research Triangle Park, NC 27709, U.S.A. 0300-483X/89/$03.50 © 1989 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

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INTRODUCTION The fungitoxic action of the broad spectrum fungicide, benomyl [methyl-1(butylcarbamoyl)-2-benzimidazole carbamate] is believed to reside in the metabolite carbendazim (MBC, methyl 2-benzimidazole carbamate) [1,2]. A number of studies have shown that MBC and benomyl (presumably through conversion to MBC [3]) are able to damage the male reproductive system (e.g., [4--7]. Significant decreases in fertility, along with declines in testicular weight, epididymal sperm counts and vas deferens sperm concentrations have all been observed [5,8,9]. In contrast, the few reproductive endocrine measures that have been included in such studies, such as serum testosterone and serum gonadotropins (luteinizing hormone and follicle-stimulating hormone) were reported to be unaltered statistically. In order to test the possibility that extragonadal endocrine influences do contribute to the adverse effects of MBC on reproductive activity, the present study focuses more specifically on the brain and pituitary components of the male reproductive axis following subchronic exposure to increasing doses of carbendazim. MATERIALS AND METHODS Forty-five timed-pregnant Long-Evans hooded female rats [Crh(LE)BR, Charles River Laboratories, Portage, MI] were purchased on day 13 of gestation and housed on a 12 h :12 h light/dark cycle (lights on 0600 h) with food and water ad libitum. After birth, litters were culled to 8 pups (4 males/ 4 females). At 3 weeks, the pups were weighed, weight-ranked by sex and weaned in unisexual pairs. Males were assigned to treatment groups that were initially equated for means and variances in body weight. Beginning on day 21, the animals were gavaged daily for 85 days with a suspension of MBC (Dupont, 98.1°/o purity) in corn oil in doses of 50, 100, 200 or 400 mg/kg body weight. Sham controls received vehicle only. Fertility was assessed by pairing treated males with females for 20 days beginning on day 85. Failure to produce viable offspring following this period of pairing (which encompassed 5 normal 4-day estrous cycles) was considered as indication of infertility. Twenty-four hours following the final MBC administration, the rats were killed within 15 s of removal from their home cages. Trunk blood was collected for serum hormonal analysis of luteinizing hormone (LH), folliclestimulating hormone (FSH), prolactin (Prl) and thyroid-stimulating hormone (TSH). Brains and pituitaries were promptly removed. Anterior pituitaries were separated from the neurointermediate lobes and weighed, before being sonicated (Fisher Model 300 sonic dismembrator) in HEPES-buffered (10 mM) Medium 199/0.3% bovine serum albumin (pH 7.4) and frozen at - 6 0 ° C . For brain tissue, the hypothalamus was sectioned into the anterior region [containing the cell bodies for gonadotropin-releasing hormone (GnRH)] and the mediobasal region (containing the axon terminals from which GnRH is

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secreted into the portal blood). The mediobasal hypothalamus (MBH) was defined rostrally by a cut at the ventral margin of the optic chiasm, caudally by a cut immediately behind the infundibulum, laterally by 2 cuts 2 mm medial to the hypothalamic sulci, and dorsally by a cut at the base of the anterior commissure. The anterior hypothalamic region (AH) was defined as that tissue overlying the rostral-to-caudal margins of the optic chiasm and with the same dorsal and lateral parameters as those used for the MBH. Individual MBH and AH regions were weighed and sonicated in 0.1 N HC1 (1:10 w/v) and frozen at - 6 0 ° C . Means and standard errors of tissue weights (in milligrams) from these areas for each of the treatment groups as are follows: AH -- 13.11 _* 0.72 (Controls), 12.58 _+ 0.68 (50), 12.50 _+ 0.62 (100), 11.96 __ 0.93 (200), 12.00 _+ 1.07 (400) and MBH -- 14.82 _+ 0.73 (Controls), 13.95 __ 0.51 (50), 12.42 _+ 0.73 (100), 13.79 _+ 1.07 (200), 13.63 _+ 0.43 (400). For analyses of GnRH, thawed samples were centrifuged (30 min, 3000 rev./min), and 20 ~1 of supernatant (pH adjusted to 7.4) was aliquoted into assay tubes. H o r m o n e assays

All hormones were analyzed by radioimmunoassay. The concentrations of LH, FSH, Prl and TSH were determined with the following materials kindly provided by the National Institute of Arthritis, Diabetes and Digestive and Kidney Diseases: Iodination preparation -- I-6 (LH), I-6 (FSH), I-5 (Prl), I-7 (TSH); Reference prep. -- RP-2 (LH), RP-2 (FSH), RP-3 (Prl), RP-2 (TSH); and antisera -- S-8 (LH), S-11 (FSH), S-9 (Prl), S-5 (TSH). Individual tracers were radiolaheled with 125I (New England Nuclear), using chloramine-T [10]. Each labeled hormone was separated from unreacted iodide by gel filtration chromatography as described previously [11]. The assays were carried out according to recommendations accompanying each kit, and all used goat antirabbit gamma-globulin (Calbiochem) as second antibody. Assay sensitivities for LH, FSH and TSH were increased by a 24-h co-incubation of sample and first antibody prior to the addition of labeled hormone for an additional 24 h. Inter- and intra-assay variances, respectively, were: 5.8%, 4.80/o (LH); 6.00/o, 6.20/o (FSH); 7.00/o, 5.3o/o (Prl); and 6.00/o, 4.1°/o (TSH). Assay sensitivities were: 18 pg/tube (LH), 0.15 ng/tube (FSH), 35 pg/tube (Prl), and 30 pg/tube (TSHI. Anterior and mediobasal hypothalamic GnRH concentrations were measured using antiserum No. 428 (kindly provided by Dr. P.M. Conn, Univ. of Iowa) at a tube dilution of 1:28 000. Purified GnRH for iodination was obtained from the National Hormone and Pituitary Program. It was radiolabeled by a modification of the chloramine-T procedure [10] and purified by ion-exchange chromatography on a carboxymethylcellulose column as described previously [11]. The assay was performed as detailed by Nett and Niswender [12], using second antibody to separate free hormone from bound. As with LH, FSH, and TSH, the sensitivity was optimized (1.5 pg/tube) by a prior 24-h co-incubation of sample and first antibody. The interand intra-assay variances were 7.1O/o and 5.6%, respectively.

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Statistical analysis Significant effects among groups were tested by regression analysis under the General Linear Models procedure of the Statistical Analysis System (SAS) package, with the alpha level set at 0.05. Comparisons among individual doses of MBC were further examined by Tukey's studentized range test. RESULTS

The weights for whole pituitary and anterior lobes are listed in Table I. No differences were present for these 2 measures, indicating that MBC did not affect the size of either anterior or neurointermediate regions at any of the doses employed. Figure 1 presents the serum concentrations for LH, FSH, Prl and TSH. Linear regression analysis shows that there was a dose-related increase in serum FSH (P < 0.01) which was first observed at a dose of 200 mg/kg. By 400 mg/kg, this elevation was significantly different from controls (P < 0.05). No other statistical effects were present for the other 3 serum hormones, although there did appear to be a decline in prolactin throughout the dose range. For the pituitary hormone concentrations (Fig. 2), LH showed a rise from 100 to 400 mg/kg MBC (P < 0.01). As with serum FSH, pituitary LH was significantly increased for the highest MBC group (P < 0.05). No differences in pituitary FSH, Prl or TSH were present for any of the 4 doses. The 200 and 400 mg/kg groups were then partitioned into fertile and nonfertile subgroupings and further evaluated for both gonadotropins by analysis of variance. No non-fertile rats were present in the either the 50 or 100 mg/kg groups, and consequently these groups were not included in such analyses. Figure 3 shows that pituitary LH elevations were present in the highest dose group, regardless of fertility status (P ~< 0.05, Fertile; P ~ 0.01, Non-fertile). In serum, no significant differences from controls were found, although a discrepancy was seen at 200 mg/kg between the fertile and nonfertile groups. For serum FSH, there were clear increases at both doses only TABLE I E F F E C T OF MBC ON P I T U I T A R Y WEIGHT Milligrams ± Standard Error of the Mean Control n: 12

50 mg/kg 8

100 mg/kg 8

200 mg/kg 8

400 mg/kg 7

Total pituitary weight

9.41 ± 0.32

9.93 ± 0.50

9.51 ± 0.29

9.74 ± 0.41

9.66 ± 0.31

Anterior pituitary

7.93 ± 0.31

8.34 ± 0.53

8.11 ± 0.30

8.39 ± 0.41

8.20 ± 0.31

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Fig. 1. M e a n s e r u m h o r m o n e c o n c e n t r a t i o n s following e x p o s u r e to MBC. All v a l u e s a r e in ng/ml s e r u m _ s t a n d a r d e r r o r s of t h e m e a n (S.E.M.). Group sizes a r e included with t h e L H d a t a at t h e b a s e of each column. *P < 0.05 for c o m p a r i s o n s a g a i n s t controls. 0.6

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Fig. 2. M e a n a n t e r i o r (AP) p i t u i t a r y h o r m o n e c o n c e n t r a t i o n s (in ~ g / m g tissue) following e x p o s u r e to MBC. Group sizes and v a r i a n c e s a r e depicted as in Fig. 1. *P < 0.05 for c o m p a r i s o n s a g a i n s t controls.

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Fig. 3. Mean serum and pituitary concentrations of LH and FSH at 200 and 400 mg/kg MBC for rats grouped according to fertility status. Control means are depicted as a horizontal line bisecting a shaded area representing the control S.E.M. The ratio of non-fertile animals to the group total is presented above the bars for serum LH. *P ~ 0.05, **P ~ 0.01 for comparisons against controls. 30

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for the non-fertile animals (P < 0.05, 200 mg/kg; (P < 0.01, 400 mg/kg), an effect which was not present for the pituitary comparisons. Similar partitioning of the measures for prolactin and TSH (data not shown) exhibited no such differences, nor did the values (reported by Rehnberg et al. [13]) for serum testosterone. Analyses of hypothalamic GnRH (Fig. 4) show that for the anterior region the lowest MBC dose of 50 mg/kg caused a marked elevation (P < 0.01) in the concentration of this releasing hormone as compared to controls. With increasing levels of the pesticide, there was a progressive fall in GnRH until at 400 mg/kg a significant decrease Ov < 0.05) was present. For the mediobasal region, there were no statistically significant effects of MBC, even though a gradual decline in GnRH was detectable between 100 and 400 mg/ kg. Partitioning the data by fertility status had no influence on these results (data not shown). DISCUSSION The observations of Parvinen and Kormano [4], Barnes et al. [5], and Carter et al. [8] have all implied that the adverse effect of MBC on fertility in male rats is directly on the testes. Indeed, the data from our laboratory [7,13] do indicate that gonadal tissue is a principal target site, and that such changes are present in the absence of any treatment-related shifts in body weight. However, the endocrine alterations present in hypothalamus and pituitary suggest that supragonadal changes accompany and may contribute to the reproductive failure. The disparate influence of MBC on LH and FSH underscore the involvement of differential regulatory mechanisms for these 2 gonadotropins, even though both have been reported to co-exist within the great majority of pituitary gonadotropins [14,15]. The increase in pituitary LH in the absence of a change in serum concentration suggests an augmentation by MBC in the relative rates of LH synthesis (or possibly a decline in catabolism). Since the synthesis and release of LH appear to be processes independently regulated by GnRH [16], it is conceivable that alterations in GnRH release at the higher doses may differentially affect serum and pituitary LH. The presence in the hypothalamus of a distinct FSH-releasing hormone that functions independently of GnRH has been postulated [17,18]. Accordingly, it may be that the increase in serum FSH seen at the higher doses of MBC is associated with a central and differential effect on separate LH and FSH releasing factors. The increase in serum FSH may, on the other hand, be attributable to an MBC-induced change at a gonadal level. Testosterone has been found to have inhibitory feedback effects on both LH and FSH [19,20]. However, under the present conditions, serum testosterone was not different than controls [13], even when groups were partitioned according to fertility status. Rehnberg et al. [13] did find that 200 and 400 mg/kg MBC elevated androgen binding protein (ABP) concentrations in serum and seminiferous tubules. Since this

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binding protein has been considered to be a useful index of Sertoli Cell effects [21,22], it is possible that there is an alteration in the secretion of the Sertoli cell hormone, inhibin, whose inhibitory feedback influence on pituitary FSH release is well established [23-25]. The differential effects, then, seen for LH and FSH may actually be attributable to an increase in secretion of FSH associated with diminished levels of circulating inhibin over the course of the dosing period. However, for this to be true, the effect of MBC on Sertoli cell secretory activity would have to be more complex than a straightforward reduction in release, since there would exist an inverse relationship between the release of inhibin and ABP. What is clear is that there is a relationship between those changes observed in FSH and fertility. By 200 and 400 mg/kg MBC, there is an increase in the number of non-fertile males [7] for which serum FSH is significantly elevated. Such an effect could be associated with an above-mentioned alteration in inhibin release, although a definitive relationship remains to be established. The effect of MBC on anterior hypothalamic concentrations of GnRH is difficult to interpret. The cell bodies for the GnRH neurons that project their axons into the median eminence region of the MBC reside in the anterior area. It may be that low doses of MBC stimulate GnRH synthesis and/or suppress the axoplasmic transport of this hormone to the neuronal terminals, something which may not be reflected in MBH values, given the large differences in regional concentrations. Increasing amounts of the fungicide may then cause a dose-related decline in GnRH, which by 400 mg/ kg could be reflected in the decline in MBH concentrations. It may also be that the fall in GnRH at this high dose may reflect an increase in GnRH release into the portal blood, contributing to the augmentation in both serum LH and FSH for non-fertile animals when compared to fertile ones. However, the absence of a fertile/non-fertile difference in GnRH concentrations makes this interprepation unlikely. While the particular mechanism(s) underlying these changes in the hypothalamic-pituitary-gonadal axis is unclear, the benzimidazoles have been shown to bind to the sensitive microtubule system within the cell [26,27]. Since the microtubules are implicated in a variety of functions, including cell motility, ciliary and flagellar movement, chromosome movement, maintenance of cell shape and form, intracellular and axoplasmic transport, and the anchorage of cell surface receptors [28], it would not be surprising that interference with such functions could lead to a number of perturbations in reproductive physiology. It has already been reported [29-31] that colchicine-induced disruptions of microtubular structure within testicular Leydig cells, ovarian (follicular) granulosa cells, or adrenal cell preparations are able to augment steroidogenesis. Rehnberg et al. [13] report a similar effect following in vivo dosing with MBC on hCG-stimulated testosterone release in vitro from decapsulated testes. Thus, one possible explanation for the observed effects on anterior hypothalamic GnRH, as mentioned previously, is a disruption in axoplasmic transport associated with an MBCinduced microtubular depolymerization. 180

In s u m m a r y , the p i t u i t a r y a l t e r a t i o n s o b s e r v e d in the c u r r e n t s t u d y t h a t are p r e s e n t following s u b c h r o n i c dosing with the fungicide c a r b e n d a z i m are associated with parallel c h a n g e s in fertility. It a p p e a r s likely t h a t such hormonal effects involve c o n c o m i t a n t shifts in the n o r m a l feedback from the t e s t e s t h a t are i n d e p e n d e n t of t e s t o s t e r o n e and are possibly linked to alterations in Sertoli cell-pituitary signaling. The o b s e r v e d changes in a n t e r i o r h y p o t h a l a m i c G n R H are a p p a r e n t l y u n r e l a t e d to fertility and m a y involve a direct effect on the central n e r v o u s s y s t e m , one t h a t is p r e s e n t a p a r t from the changes in gonadal and p i t u i t a r y - g o n a d a l functioning. Finally, while t h e r e is some information c o n c e r n i n g the action of MBC at a cellular level, the specific mechanism(s) u n d e r l y i n g t h e s e endocrine shifts in the r e p r o d u c t i v e s y s t e m r e m a i n to be d e t e r m i n e d . ACKNOWLEDGMENTS The r e s e a r c h described in this article has been r e v i e w e d by the Health Effects R e s e a r c h L a b o r a t o r y , U.S. E n v i r o n m e n t a l P r o t e c t i o n A g e n c y and has been a p p r o v e d for publication. A p p r o v a l does not necessarily signify t h a t the c o n t e n t s reflect the views and policies of the A g e n c y , nor does m e n t i o n of t r a d e n a m e s or commercial p r o d u c t s c o n s t i t u t e e n d o r s e m e n t or r e c o m m e n d a t i o n for use. The a u t h o r s e x t e n d a p p r e c i a t i o n to J o s e p h O s t b y and J a n e t Ferrell for their excellent technical assistance and to Drs. J o h n L a s k e y and Susan L a w s for helpful editorial c o m m e n t s . We also wish to t h a n k the National I n s t i t u t e of A r t h r i t i s , Diabetes, and Digestive and K i d n e y Diseases and the National H o r m o n e and P i t u i t a r y P r o g r a m for the gift of h o r m o n e r a d i o i m m u n o a s s a y materials. REFERENCES 1

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