Neurotoxic esterase in rooster testis

77, 175-180(1985)

TOXlCOLOCYANDAPPLlEDPHARMACOLOCiY

Neurotoxic MARCELLO

LoTTI,*‘~.~

*The

California tSchoo1

Northern

Esterase in Rooster Testis’

EDDIE T. WEI,? ROBERT C. SPEAR,? AND CHARLES

Occupational Health Center, University of Public Health, University of Cal~ornia,

Received

April

of California, San Francisco, Berkeley, Cal~ornia 94720

26, 1983; accepted August

E. BECKER* California;

and

22, I984

Neurotoxic Esterase in Rooster Testis. Lorn, M., WEI, E. T., SPEAR, R. C., AND BECKER, E. ( 1985). Toxicol. Appl. Pharmacol. 77, I75- 180. Neurotoxic esterase (NTE) is the putative target protein in the nervous system for the initiation of organophosphorus-induced delayed neuropathy. Here it is reported that NTE activity is present in rooster testis. Complete titration of rooster testis phenyl valerate esterases with paraoxon shows that about 15% of the enzymic activity is resistant to paraoxon. NTE activity after complete mipafox titration accounts for 30% of paraoxon-resistant phenyl valerate esterasesand corresponds to 7.93 f 0.39 nmol/min/ mg of protein (i f SD, n = 7). Testis NTE is inhibited in vitro similarly to brain NTE by several organophosphorus compounds. Subcellular fractionation studies of the testis indicate that most NTE activity is particle bound. Testis NTE is also inhibited in vivo by several organophosphorus esters but to a lesser extent than brain NTE. Birds dosed with organophosphorus compounds, causing delayed neuropathy, became grossly ataxic, but no testicular pathology was noted by light microscopy in roosters killed 15 days after administration. Serum testosterone levels also measured 15 days after dosing were not different from those of a control group. Recovery of NTE activity was faster in testis than in brain (4 days vs 6 days to recover to 50% of initial activity) in animals that received a high dose of an organophosphorus ester which cause delayed neuropathy. 0 1985 Academic PRSS. hc. C.

A number of organophosphorus esters (OPs) cause delayed neuropathy (OPIDN) in the hen and other sensitive species. Neurotoxic esterase(NTE), a protein found in the nervous tissue, has been characterized as the target and the mechanism of this toxicity has been reviewed (Johnson, 1982). Recently the distribution of NTE in nonneuronal tissues ’ Presented in part at the 22nd Annual Conference of the Society of Toxicology, Las Vegas, Nev., March 711, 1983. * Department of Medicine, University of California, San Francisco General Medical Center, Building 30, 5th Floor, San Francisco, Calif. 94110. 3 Present address and to whom correspondence should be sent: Istituto di Medicina de1 Iavoro dell’Universim di Padova, Via Facciolati 71,35127 Padova, Italy.

of the adult hen has been investigated, and it has been shown that NTE activity was present in the lymphatic tissues(Dudek and Richardson, 1982). Some NTE activity was also detected in the heart but the source of this activity may be from the synaptic junctions of the autonomic nervous system. A study of Huang et al. (1979) prompted our interest in roosters. They reported that the OP herbicide Amiprophos (O-ethyl 0-4methyl - 6 - nitrophenyl - N- isopropylphosphoroamidothionate) produced OPIDN in roosters, but, in addition, testicular toxicity was noted. We decided to look for NTE activity in rooster testis and to determine if inhibition of this enzyme was associated with organ damage.

175

0041-008X/85 Copyright All

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8

1985

of reproduction

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form

rmrwd.

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LOTTI

METHODS Inhibitors. Mono-o-cresyldiphenyl phosphate (MOCP) was obtained from Coalite Chemical Company, Chesterfield, Derbyshire, England. Diisopropylphosphorofluoridate (DFP) and phenylmethanesulphonyl fluoride (PMSF) were purchased from Sigma Chemical Company. St. Louis, Missouri. Purified paraoxon (diethyl-p-nitrophenyl phosphate) and mipafox (iV,N’diisopropylphosphorodiamino fluoridate) were gifts of M. K. Johnson (MRC Carshalton, United Kingdom). Analytical grade methamidophos was a gift of Chevron Chemical Company, Richmond, California. Biochemical assays. The assay for NTE is based on the differential sensitivity of phenyl valerate (PV) e&erases in brain homogenate to inhibition by neurotoxic and nonneurotoxic OPs as described originally by Johnson (1975). Preincubation with paraoxon, a nonneurotoxic compound, inhibits most of the total brain PV esterase activity. When mipafox, a neurotoxic inhibitor, is included in the preincubation medium, an additional PV activity is inhibited. This additional decrease in enzymic activity represents NTE activity. To define testicular NTE we used the complete titration curves (over a wide range of concentrations) with the neurotoxic and nonneurotoxic inhibitors of rooster testis PV esterase activity as previously described (Dudek and Richardson, 1982). All other NTE assays were performed by a standard method (Johnson, 1977), but using twice the tissue concentration (about 12 mg of tissue, wet weight). Protein contents were measured according to Lowry et al. (195 1). In vitro studies. Testes were removed from three birds, immediately homogenized, and pooled for inhibition studies. The concentration for 50% inhibition (150) of enzyme by OPs were obtained from plots of log activity remaining versus inhibitor concentration (Lotti and Johnson, 1978). The mipafox I50 was calculated after subtraction of the residual activity which is resistant to high concentrations of the inhibitor. Subcellular fractions were obtained by differential centrifugation (De Duve et al., 1955). A bovine brain microtubular preparation, obtained by an assembly-disassembly method (Asnes and Wilson, 1979), was a generous gift of Professor Leslie Wilson, University of California, Santa Barbara, California. In vivo studies. Random bred adult red roosters (Gallus domesticus weighing 2.5 to 4.0 kg) were caged three per group and allowed feed and water ad libitum. Paired groups of animals were injected with several organophosphorus esters (Table 2). One group of animals was killed 24 hr after dosing; the brain and testes were immediately dissected, washed in ice-cold 50 mM TrisHCl buffer (pH 8.00 at 23°C) containing 0.2 mM EDTA, homogenized (10% w/v) with a Polytron homogenizer, and tested for NTE activity. The other group was killed 15 days after OP dosing; the testes were removed im-

ET AL. mediately, bisected longitudinally, fixed in 10% buffered Formalin, embedded in paraffin, and stained with hematoxylin-eosin for histology. Blood collected from the wing vein at the time the animals were killed was allowed to stand at room temperture for 1 to 2 hr. Serum was then separated and frozen at -80°C until assayed for testosterone. The serum testosterone radioimmunoassay was performed according to Battke et al. (1973). Reconstituted ether extracts of serum were incubated with [ 1,2,6,7-‘HItestosterone antiserum (Wien Laboratories, Succasunna, N.J.). The supematant fraction was transferred to scintillation vials containing 10 ml scintillant (scintillation grade toluene containing 0.1% 2.5-diphenyloxazole and 0.0 1% 1,4-bis-2-(5-phenyloxazolyl)benzene). Serum testosterone concentration was calculated from a testosterone reference curve (Candrosten-17j3-o1-3-one, Sigma Chemical Co.). Intraassay variation was 6%. To study the rate of reappearance in rooster testis, animals which had received a single dose of MOCP (70 mg/kg, po) were killed at different times after dosing, organs were dissected, and NTE was assayedas described above. No changes in feed intake were observed in dosed birds during this experiment.

RESULTS Complete paraoxon and mipafox titrations of rooster testis PV esterases are shown in Figs. 1 and 2. Figure 1 shows that 10% of all PV esterases of rooster testis are resistant to high concentrations of paraoxon. Figure 2 shows that NTE accounted for about 25 to 30% of this paraoxon-resistant PV esterase activity. The specific activity of rooster testis NTE was 7.39 + 0.39 nmol/min/mg of protein (X + SD, y1= 7). 150’s for rooster testis NTE in vitro were also calculated for several inhibitors: NTE was inhibited for 20 min at pH 8.0 at 37°C and the 150’s, calculated according to Lotti and Johnson (1978), are reported in Table 1. The 150’s for brain and testes were within a factor of 2, suggesting that the two enzymes are the same with respect to inhibition by these compounds. Crude subcelhtlar h-actions of rooster testis homogenates showed that the majority of NTE activity (>90% of the whole homogenate activity) was particle bound.

ROOSTER TESTIS NEUROTOXIC

177

ESTERASE TABLE 1

IN VITRO INHIBITION OF NTE BY SOME OP ESTERS: COMPARISON BETWEEN TESTIS AND BRAIN ENZYMES NTE 150 (mM)’

FIG. 1. Effect of increasing concentration of paraoxon on hydrolysis of phenyl valerate by rooster testis homogenate. Preincubation was performed at 37°C for 20 min, and the hydrolysis of substrate at 37°C for 15 min. Each point is X + SD from three homogenates, each from one bird.

We hypothesized that NTE may be found in microtubuli because such organelles are common to brain and testis and biochemically very similar. However, only traces of NTE were detected in a preparation of bovine brain microtubules.

Inhibitor

Testis

Brain

Diisopropylphosphorofluoridate (DFP) N,N-Diisopropylphosphorodiamidofluoridate (mipafox) Phenylmethanesulphonyl fluoride (PMSF) O,S-Dimethylphosphoroamidothioate (methamidophos)

0.0035

0.004

0.013

0.007

0.05

0.06

2

1.2

“NTE was inhibited for 20 min at pH 8.0, 37°C; the values were calculated according to Lotti and Johnson (1978).

Table 2 shows the comparative inhibition of brain and testis NTE in vivo and the neurological assessment of birds. With all the inhibitors (DFP, PMSF, and MOCP), the inhibition of testis NTE was less than that of the brain. Similarly dosed birds, killed 15 days after dosing, were paralyzed if dosed with an OP which causes OPIDN, but no histological signs of toxicity were found in the testes. Serum testosterone in the treated birds was not significantly different from a control group, when measured at the time when birds were killed. The NTE activity recovered faster in the testis than in the brain of roosters dosed with MOCP (Fig. 3). DISCUSSION

FIG. 2. Effect of increasing concentrations of Mipafox on the hydrolysis of phenyl valerate by paraoxon-resistant phenyl valerate exterases of rooster testis homogenate. Same conditions as Fig. 1.

The results demonstrate the presence of NTE in rooster testis. The significance of NTE in the testis is not known, but we have attempted to determine if the inhibition of NTE in testis is associated with tissue damage. According to our results, brain and testis NTE inhibition were not the same, testis being inhibited less. Because the 150’s in vitro of several OPs are the same for both NTEs, these results suggest that accessibility of OPs to the testicular enzyme is more difficult. OPs which cause OPIDN inhibit neural NTE at about 70 to 80%, which is considered the

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LOTTI

ET AL.

TABLE 2 IN

VIVO INHIBITION

OF BRAIN

AND

TESTIS

NTE

IN ROOSTERS

90 NTE inhibition“ Brain

Testis

Grade of ataxiah

0.8, SC 2.4. SC

21.11 69, 67. 68

0, 0 37, 11, 19

2. 1.2

30, SC

68, 80. 74

40, 60, 49

0, 0, 0

70, PO 14O.P 250, po

95, 94. 94 99.98 99, 100

65, 61, 76 84, 90 93,94

3, 3, 4 4, 4 4, 4

Dose @g/kg), route

Inhibitor Diisopropylphosphorofluoridate

(DFP)

Phenylmethanesulphonyl fluoride (PMSF) Mono-2-cresyl-diphenyl

phosphate (MOCP)

0. 0

’ Measured 24 hr after dosing. b Ataxia was essessedon Day I5 after dosing, according to the 4-point scale of Johnson and Barnes (I 970). First, second, and third values for both NTE inhibition and grade of ataxia, are from birds 1,2, and 3, respectively.

da!s 1

2

3

I

E

FIG. 3. Comparison of rates of return of NTE activity in brain (A) and testis (0). Animals received a single dose of MGCP 70 mg&, po in corn oil (0.6 to 0.8 ml). Each point represents the NTE values for an individual animal. Day 0 is 24 hr after dosing. The inhibition was about 94% for brain NTE and 76% for testis NTE. For calculations the inhibition at 24 hr was considered 100%. Each line is the best computed line (least square) through the experimental points. The slopes of the two lines differ significantly (Student’s t test, p < 0.05). In one animal killed on Day 10, brain NTE was still inhibited (30%) but testis NTE was in the normal range.

threshold for developing peripheral neurop athy (Johnson, 1982). More than 90% inhibition of NTE was obtained in rooster testis with highly neurotoxic doses of MOCP, but no signs of histological damage were found 15 days later. However, a number of factors might account for these negative results. The time of removing the testes for histology might not have been appropriate according to the spermatogenic cycle of the rooster. However, for a single dose of a neurotoxic OP, the duration of the experiment is limited by the declining health of the paralyzed birds. Histology may not be an appropriate endpoint to assess this toxicity. NTE protein may not have the same physiological importance in the testis that it has in the nervous system. The higher speed of reappearance of NTE in the testis in comparison to the brain might represent a higher rate of protein synthesis and possibly a more effective mechanism of repair. Limited data are available on the testicular toxicity of OPs (Lucier et al., 1977). Dimethyldichlorovinyl phosphate (DDVP), a weakly neurotoxic OP (Caroldi and Lotti, 198 I), was reported to cause alterations to the seminiferous epithelium and the Leydig cells of the rat after po administration (Krause and Homola, 1974). Dimethylmethyl phos-

ROOSTER

TESTIS NEUROTOXIC

phonate, a poor esterase inhibitor, causes altered reproduction function, which is dose related. Histologic abnormalities of the testis were seen only at very high doses (Dunnick et al., 1984). The OP herbicide Amiprophos, reported as toxic to the testis by Huang et al. (1979), is also a potent inhibitor of tubulin synthesis (Kiermayer and Fedtke, 1977; Collis and Weeks, 1978). This effect on microtubules, however, does not seem related to the ability of the OP to inhibit esterases because it is caused in vitro in the absence of a metabolic activation system, by P=S OPs which are not good esterase inhibitors. Furthermore, NTE activity was not found in microtubule preparations from cows, a species susceptible to OPIDN (Johnson, 1982). While the mechanism of the testicular toxicity of Amiprophos is unknown, the neurotoxicity is related to the phosphorylation of neurotoxic esterase. Some toxic neuropathies, for example, the neuropathy caused by Vinca alkaloids, have been associated with microtubular damage (Sterman and Schaumburg, 1980), and a possible role of microtubuli in OPIDN has been suggested (Seifert and Casida, 1982). Several chemicals which cause peripheral neuropathy due to axonal degeneration are toxic to the testis, including n-hexane, and its toxic metabolites 2,5hexanedione and 2hexanone (Krasavage et al., 1980), acrylamide (Hashimoto et al., 1981), Vinca alkaloids (Dixon, 1980) and pbromophenylacetylurea (Cavanagh, 1973). Although a recent paper suggested that altered lipid metabolism could be the link between 2,5-hexanedione-induced testicular atrophy and peripheral neuropathy in rats (Gillies et al., 198 l), these changes could be secondary to testicular atrophy. We have reported that testicular NTE can be significantly inhibited, yet the histology of the testis remains unaffected. Thus it is unlikely that acute inhibition of testicular NTE plays a causal role in the testicular toxicity seen after Amiprophos. The biological significance of the presence

ESTERASE

179

of NTE in testis tissue is unknown. We know that NTE catalytic activity is not relevant to the pathogenesis of OPIDN, in fact, it is the second step after the progressive phosphorylation of the active site which causes OPIDN, the “aging” of the phosphoryl-enzyme complex (Johnson, 1982). Since NTE was also found in the lymphatic system of the hen (Dudek and Richardson, 1982) we wonder whether there is any physiological connection between these systems mediated by NTE protein; several fascinating speculations concerning connections between the nervous, hematopoietic, and germ cell systems have been suggested (Golub, 1982). ACKNOWLEDGMENTS We thank J. B. Cunningham, D. A. Seid, D. J. Goldfield, and J. Rigod for technical help and Professor Margaretten, M.D., San Francisco General Hospital, and A. Fassina, M.D., Istituto di Anatomia Patologica dell’Universiti di Padova, for histological examinations.

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