Toxicology, 76 (1992) 89-100 Elsevier Scientific Publishers Ireland Ltd.

89

Competitive studies relating to tryptophan binding to rat hepatic nuclear envelopes as a sensitive assay for unknown compounds* Herschel Sidransky, Ethel Verney and James W. Cosgrove Department of Pathology, The George Washington University Medical Center, Washington, DC 20037 (USA) (Received June 3rd, 1992; accepted July 17th, 1992)

Summary Our laboratory has reported that L-tryptophan binds to a rat liver nuclear envelope protein and this binding is saturable, stereospecific and of high affinity. Utilizing an in vitro [3H]tryptophan binding assay to hepatic nuclear envelopes, we have determined the effects of using excess unlabeled Ltryptophan from a number of different suppliers. This study reports that, based on our in vitro binding assay, some significant differences were observed when implicated L-tryptophan in cases of the eosinophilia-myalgia syndrome obtained from a Japanese manufacturer~ Showa Denko, was assayed, in contrast to non-implicated L-tryptophan from other suppliers. An isolated impurity of Showa Denko Ltryptophan, 1,1 '-ethylidenebis(tryptophan) alone or together with non-implicated L-tryptophan or its breakdown product, l-methyl-1,2,3,4-tetrahydro-fl-carboline-3-carboxylic acid, did not appreciably affect the in vitro [3H]tryptophan binding to hepatic nuclear envelopes as did the Showa Denko L-tryptophan. Our data, derived with our in vitro binding assay system, suggests that impficated L-tryptophan from Showa Denko contains a compound/s (unknown at present) other than 1,1 '-ethylidenebis(tryptophan), which alters in vitro [3H]tryptophan binding. The significance of the impurity/ies involved remains to be determined.

Key words: Liver nuclear envelopes; 1,1 '-Ethylidenebis(tryptophan); In vitro tryptophan binding assay

Introduction With the advent of the eosinophilia-myaigia syndrome, epidemiologic data have implicated L-tryptophan from a single manufacturer [1-3]. This tryptophan has been described as containing an impurity to which much significance has been attached. This impurity, peak E or peak 97 by HPLC, has been analyzed and found to be 1,1 '-ethylidenebis(tryptophan) [4,5]. Several experimental studies have been instituted to determine whether this ditryptophan compound may cause toxic effects [6,7]. *This research was supported by a research grant from Showa Denko KK. Correspondence to: Herschel Sidransky, M.D., Professor and Chairman, Department of Pathology, The George Washington University Medical Center, 2300 Eye Street, NW, Washington, DC 20037, USA. 0300-483X/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

90 For many years, our laboratory has been concerned with the unique biochemical properties of L-tryptophan, particularly on mammalian liver [8]. Earlier, we reported that L-tryptophan binds to a rat liver nuclear envelope receptor protein and that this binding is saturable, stereospecific and of high affinity [9]. A number of analogs, metabolites or related compounds of L-tryptophan have been tested and a few have been found to compete with in vitro [3H]tryptophan binding to hepatic nuclear envelopes [10,11]. In view of these earlier background studies concerning L-tryptophan binding to hepatic nuclear envelopes, we decided to utilize our sensitive in vitro assay system to explore whether we could detect possible differences when using L-tryptophan from different sources, including the L-tryptophan implicated in cases of the eosinophilia-myalgia syndrome. This study, using our in vitro binding assay, reports that some interesting differences in binding were observed when implicated Ltryptophan compared to control L-tryptophan was assayed. This effect with the implicated tryptophan occurred whether or not it contained high (146 ppm) or low (6 ppm) 1,1 ' -ethylidenebis(tryptophan). Methods

Animals Female rats of the Sprague-Dawley strain (Hilltop Lab Animals, Inc., Scottsdale, PA), average weight 175 g (range 150-250 g), were used in the experiments. The rats were maintained in a temperature-controlled room with a 12-h light/dark cycle. Before the experiments were begun, the animals were deprived of food overnight but had free access to water. These studies were approved by the institutional animal care and use committee. Chemicals The [3H]tryptophan used in the experiments was L-[5-3H]tryptophan, 1132 GBq/mmol, obtained from NEN Research Products, Du Pont (Boston, MA). [614C]orotic acid, 1.48 GBq/mmol, was obtained from the same source and L-[U14C]leucine, 12.9 GBq/mmol, was obtained from Amersham/Searle (Arlington Heights, IL). The test compounds were obtained from Sigma Chemicals (St. Louis, MO) or from US Biochemical (Cleveland, OH). In one set of experiments, Ltryptophan from several different United States suppliers was used. l , l ' ethylidenebis(tryptophan) (E) and 1-methyl- 1,2,3,4-tetrahydro-/~-carboline-3-carboxylic acid (X) were obtained from Showa Denko. Isolation of nuclei Rat hepatic nuclei were prepared as described by Blobel and Potter [12]. The liver tissue was minced and homogenized with three strokes in 2 vols. of buffer A [0.05 mol/1 Tris-HC1, pH 7.5, 0.025 mol/l KCI, 0.005 mol/1 MgCI2, 0.0001 mol/l phenylmethylsulfonyl fluoride (PMSF), 0.0002 mol/l dithiothreital and 0.25 mol/l sucrose]. The homogenate was filtered through cheesecloth (four layers) prior to mixing with 2 vols. of Buffer B (0.05 mold Tris-HCl, pH 7.5, 0.025 mol/1 KC1, 0.005 mol/1 MgC12 and 2.3 mold sucrose). The latter was underlaid with 1 vol. of buffer

91 B and centrifuged for 60 min at 105 000 x g at 4°C in a Beckman L5-75 ultracentrifuge. The supernatant was discarded, and the pellet was washed twice with buffer A before further use.

Preparation of nuclear envelopes Nuclear envelopes were isolated with the procedure of Harris and Milne [14] as modified by Agutter and Gleed [13] and is routinely used in this laboratory [9,15]. Purified hepatic nuclei were treated with 0.001 mold NaHCO3, digested with DNase and centrifuged on a stepwise sucrose gradient (up to 2 mol/1 sucrose); then the nuclear envelope band at interface (1.5-1.8 mol/l sucrose) was removed. The purified nuclear envelopes were resuspended in a binding assay buffer C (0.05 mold Tris-HC1 (pH 7.5), 0.002 mol/l EDTA, 10% v/v glycerol, 0.001 mol/l PMSF, 0.002 mol/l/3-mercaptoethanol).

Binding of [3H]tryptophan to nuclei or nuclear envelopes Rat hepatic nuclei or nuclear envelopes prepared as described above were incubated with L-[5-aH]tryptophan (containing 278 kBq and 0.245 nmol L-tryptophan/assay) in the absence or presence of a 2000-fold excess of L-tryptophan (10 -4 mol/1) or test compound (10 -4 mold) at room temperature for 2 h. This concentration was selected based upon our earlier findings [9]. The nuclei were then washed three times with buffer A and the nuclear envelopes were washed two times with buffer C to remove free and loosely bound radioactivity. After the final wash, the nuclei or nuclear envelopes were suspended in buffer A or in buffer C, respectively and then radioactivity was measured after adding a scintillation mixture (ACS II from Amersham/Searle). Binding of [3H]tryptophan to hepatic nuclei or nuclear envelopes was expressed as cpm per unit protein (total binding in absence of unlabeled Ltryptophan or test compound minus binding in presence of 2000-fold excess of unlabeled L-tryptophan or test compound). Values of test compounds were then compared with values obtained using unlabeled L-tryptophan (control group).

Preparation of polyribosomes Postmitochondrial supernatants were prepared from homogenates of livers of rats of each group and were used for size distribution analysis of polyribosomes after addition of deoxycholate as described previously [16]. The degree of polyribosomal aggregation of livers under the different experimental conditions was evaluated from the patterns obtained by sucrose density gradients. This was conducted by calculating the relative distribution of monomers-dimers in relation to total ribosomes by measuring the area under the monomer and dimer peaks and the area under the entire pattern (monomers-dimers plus the other polyribosome fractions) of each gradient pattern.

In vitro protein synthesis Microsomes prepared from postmitochondrial supernatants of livers of control and experimental rats were used for studies on incorporation in vitro as described earlier [16]. In all assays, cell saps of livers of control (distilled water-treated) rats were used. L-[U-14C]Leucine, 18.5 kBq, was added to each incubation tube.

92 Radioactivity in protein was measured using a liquid scintillation spectrometer (Beckman Instruments, Palo Alto, CA). The protein was determined as described by Lowry et al. [171. Enzyme assay Nucleoside triphosphatase (Mg2+-dependent adenosine triphosphatase, EC 3.6.1.3) (NTPase) was assayed according to the method of Agutter et al. [18]. The assay depends upon the determination of the inorganic phosphate released from the substrate (ATP) during the incubation with hepatic nuclei for 30 rain at 35°C. Statistics Data were expressed as means 4- S.E.M. to enable analysis by Student's t-test [191. Results

In order to determine whether L-tryptophan (case implicated) obtained from Showa Denko reacted differently than L-tryptophan from other sources, we conducted experiments to measure to what degree each of the tryptophans inhibited in vitro [3H]tryptophan binding to hepatic nuclei or nuclear envelopes. First, we determined whether excess unlabeled L-tryptophan obtained from a variety of United State companies reacted similarly in relation to inhibition of in vitro [3H]tryptophan binding to hepatic nuclei or nuclear envelopes. As control or reference L-tryptophan, we used L-tryptophan from United States Biochemical Corporation (USBC) in this as well as in all other experimental studies. In comparing the effects due to L-tryptophan from different chemical suppliers upon [3H]tryptophan binding in vitro in two to three experiments, the relative specific [3H]tryptophan binding to hepatic nuclei and nuclear envelopes, respectively were as follows: USBC (Standard), Lot No. 34936, 100%, 100%; Gibco Laboratories, Lot No. 11000, 105.1%, 98.7%; Gibeo Laboratories, Lot No. 73285, 100.8%, 96.9%; ICN Biomedicals, Lot No. 11177, 107.8%, 97.3%; General Biochemicals, Lot No. 541, 102.9%, 99.4%; and Schwartz-Mann, Lot No. 2876, 109.4%, 99.8%. It is apparent that with assays using excess unlabeled tryptophan (10-4M) from the different sources and hepatic nuclei or nuclear envelopes the results were basically the same. Next, we investigated the effects of using batches of L-tryptophan from Showa Denko, Lot No. 3206203 (SDA), which contained low (3 ppm) E and Lot No. 67116202 (SDB), which contained high (146 ppm) E. The results as specific binding are summarized in Table I. Specific binding (%) =

total binding - non-specific binding total binding

X

log

where non-specific binding was binding obtained in presence of 2000-fold excess of unlabeled L-tryptophan (from different sources and batches). It is apparent that the in vitro specific [3H]tryptophan bindings were similar for all three groups when using hepatic nuclei. However, when using hepatic nuclear envelopes, the specific

93 TABLE I COMPARISON OF EFFECTS DUE TO L-TRYPTOPHAN FROM DIFFERENT SOURCES UPON [3H]TRYPTOPHAN BINDING IN VITRO TO HEPATIC NUCLEI AND NUCLEAR ENVELOPES Source of tryptophan x

Specific [3H] Tryptophan bindingz (%)

USBC (No E) SDA (Low E) SDB (High E)

Nuclei

Nuclear envelopes

(7) 64.5 (7) 62.9 (7) 64.2

(20) 63.7 ± 1.55' (16) 55.7 -~ 2.77a (20) 55.5 4- 1.83b

x Unlabeled compounds added at 10.-4 M. z Specific binding (%) =

total binding - non-specific binding x 100 total binding

where non-specific binding was binding obtained in presence of 2000-fold excess of unlabeled L-tryptophan (from different sources and batches). *Number of experiments in parentheses. Means ± S.E.M. a0.01 < P < 0.02. bp < 0.01.

[3H]tryptophan b i n d i n g was significantly decreased with the S D A group (-12.6%) a n d with the SDB g r o u p (-12.9%) c o m p a r e d with the U S B C group. Next, a n u m b e r o f experiments were c o n d u c t e d using different c o n c e n t r a t i o n s (10 -1° to 10 -4 M) o f u n l a b e l e d t r y p t o p h a n s (control, S D A a n d SDB) a n d in vitro [3H]tryptophan b i n d i n g to hepatic nuclear envelopes was determined. Figure 1

100 o

90

tO

o

"6

80 70

"0 c

60

0 m

50

c

40'

.o I

30'

7

20100 0

io-

1o'-s io'-,

io'-,

i

Io"

Tryptophan (M) Fig. l. Effect of increasing concentrations of unlabeled L-tryptophan (control, USBC; Showa Denko A, SDA; and Showa Denko B, SDB) on in vitro [3H]tryptophan binding to rat hepatic nuclear envelopes. Number of experiments are given adjacent to each point.

94

reveals the results. It appears that at most concentrations (10 -s to 10-4 M) of unlabeled tryptophans, the SDA and SDB tryptophans caused less inhibition of [3H]tryptophan binding to hepatic nuclear envelopes than did the control tryptophan. Though the differences at each time interval from 10-s to 10-4 M of unlabeled tryptophan were not statistically significant, probably due to variations in a small group of experiments, the differences at 10 -4 M as indicated in Table I were significant in comparing control with SDA or SDB tryptophans. Using data derived from Fig. 1, it is possible to determine the ICso (50% inhibition of specific [3H]tryptophan binding to hepatic nuclear envelopes) values for the different tryptophans. These values were as follows: control tryptophan, 3.7 × 10-9 M; SDA (low E) tryptophan, 2.5 x 10-7 M; and SDB (high E) tryptophan, 2.5 x 10-7 M. They indicate that 68-fold more SDA or SDB tryptophan is needed than control tryptophan to obtain the 50% inhibitory binding effect. Given that SDA and SDB tryptophan modifies in vitro tryptophan binding to hepatic nuclear envelopes, we investigated whether the administration of these compounds in vivo would influence the subsequent in vitro [3H]tryptophan binding to hepatic nuclear envelopes. Rats were tube-fed tryptophan (30 mg in 3 ml water/ 100 g body wt) from the three different batches (control (USBC), SDA and SDB) 10 min before killing and then the isolated hepatic nuclear envelopes were used for assay. The results indicated that the rats tube-fed the three different tryptophans revealed marked decreases in in vitro specific [3H]tryptophan binding to hepatic nuclear envelopes. The results are summarized in Table II. The experimental tryptophan (SDA and SDB) revealed a somewhat higher specific tryptophan binding (+5.2 and +6.8%, respectively) than did the control (USBC) tryptophan. In order to determine whether the effect of tube-feeding L-tryptophan from the different batches and sources for 10 min on in vitro [3H]tryptophan binding to hepatic nuclear envelopes would persist over time, we conducted several experiments

T A B L E II C O M P A R I S O N O F SPECIFIC B I N D I N G O F [ 3 H ] T R Y P T O P H A N T O H E P A T I C N U C L E A R ENVELOPES IN VITRO A F T E R T U B E - F E E D I N G T R Y P T O P H A N F R O M D I F F E R E N T SOURCES 10 rain B E F O R E K I L L I N G Group a

Specific binding, counts/min per rag nuclear envelope protein

% Change

(%)

Control (Water) T R P (USBC - - No E) T R P (SDA - - Low E) T R P (SDB - - High E)

(5) (5) (4) (5)

61.0 26.1 31.3 32.9

± ± ± ±

7.9 b 2.3 c 6.3 d 3.1 d

-57.2 -48.7 -46.1

a All rats were tube-fed water or tryptophan (TRP) (30 mg/100 g body wt) 10 min before killing. b Number of experiments in parentheses. Means ± S.E.M. c p < 0.01. d0.01 < P < 0.05.

95

where rats were killed after 1, 4 or 8 h following the tube-feedings of the standard (USBC), experimental (SDB) tryptophan, or water. No food was offered during the intervals. The results are summarized in Table III. It appears that with time the differences observed after 10 min (Table II) between standard and SDB tryptophan change such that it decreases after 1 h, reverses in effect after 4 h and becomes similar after 8 h. Also, the results indicate that the effect in vivo (tube-feeding) of a high dose of L-tryptophan (30 mg/100 g body wt) persists for many (up to 8) hours. In order to determine whether impurities in SDB tryptophan affected control tryptophan, we intermixed control (USBC) and SDB tryptophans at three concentrations (10 -4 to 10-8 M) and determined how these mixtures compared with the single components in inhibiting in vitro [3H]tryptophan binding to hepatic nuclear envelopes. The results of three to four experiments revealed that the intermixing of the two tryptophans at the three concentrations revealed similar inhibitory effects on specific binding as produced by SDB tryptophan alone. Thus, the components of SDB tryptophan at low concentrations (10 -6 or 10-8 M) are still able to affect control tryptophan in its inhibitory binding effect. In view of the preceding findings that SDB tryptophan affects [3H]tryptophan binding to hepatic nuclear envelopes, we investigated whether the component, 1,1 'ethylidenebis(tryptophan) (E), present in SDB tryptophan [3-5] may be responsible. Table IV summarizes the results of experiments testing whether 1,1 '-ethylidenebis(tryptophan) alone or together with control tryptophan affects in vitro [3H]tryptophan binding. Using excess (2000-fold) unlabeled 1,1'-ethylidenebis(tryptophan) alone did not appreciably inhibit in vitro [3H]tryptophan binding to hepatic nuclear envelopes. Inhibition of binding with excess control tryptophan plus 1,1 '-ethylidenebis(tryptophan) was not statistically different from inhibition with control tryptophan alone. Next, we investigated whether a breakdown product of 1,1 '-2 ethylidenebis(tryptophan) would affect in vitro [3H]tryptophan binding. The breakdown compound

TABLE III C O M P A R I S O N O F SPECIFIC B I N D I N G OF [3H]TRYPTOPHAN TO H E P A T I C N U C L E A R ENVELOPES IN VITRO A F T E R T U B E - F E E D I N G T R Y P T O P H A N F R O M D I F F E R E N T SOURCES 1, 4 A N D 8 h B E F O R E K I L L I N G Groups x

C T R P (USBC) T R P (SDB)

Specific binding, counts/min per m g nuclear envelope protein lh

4h

8h

(%)

(%)

(%)

(5) 67.0 a- 9.3* (5) 27.8 ± 2.2 a (5) 29.5 a- 8.2 b

(3) 64.4 ± 15.8 (3) 24.0 ± 3.9 (3) 18.5 ± 2.7 b

(3) 61.7 ± 21.6 (3) 23.1 ± 1.6 (3) 24.2 q- 2.9

XAll rats were tube-fed tryptophan (TRP) (30 mg/100 g body wt) or water (C). * Number of experiments in parentheses. Means ± S.E.M. a p < 0.01. b0.01 < P < 0.05.

96 TABLE IV INHIBITION O F TOTAL IN VITRO [3H]TRYPTOPHAN BINDING USING 2000-FOLD EXCESS OF UNLABELED TEST COMPOUNDS SINGLY OR COMBINED Compound*

TRP E TRP + E

Hepatic nuclear envelopes (%)

Change (%)

(8) 68.4 4- 3 . 2 a (8) 18.6 4- 3.5 b (3) 56.2 4- 9.3

-72.8 -17.8

* TRP (USBC tryptophan), E (1,1 '-ethylidenebis(tryptophan)); unlabeled compounds were added at 10-4 M singly or combined. a Number of experiments in parentheses. Mean ± S.E.M. b p < 0.01.

is 1-methyL-1,2,3,4-tetrahydro-/~ carboline-3 carboxylic acid (compound X) as previously described [201. We measured whether excess (2000-fold) compound X, or other carboline compounds would affect in vitro [3H]tryptophan binding to hepatic nuclear envelopes. The results summarized in Table V reveal that none of the carboline compounds were able to appreciably inhibit in vitro [3H]tryptophan binding. In general, these compounds at 2000-fold excess inhibited in vitro [3H]tryptophan binding to hepatic nuclear envelopes 61-86% less than did 2000-fold excess control (USBC) tryptophan. Also, in one experiment control tryptophan alone or plus added compound X were used and both revealed similar inhibition of in vitro [3H]tryptophan binding to hepatic nuclear envelopes, 72.5% and 64.7%, respectively. To determine whether SDA and SDB tryptophan in comparison to control (USBC) tryptophan had different biologic effects on rat liver, we investigated how tube-feeding single doses of each of the three tryptophans would affect the hepatic responses in regard to status of polyribosomal aggregation and protein synthesis in vitro. In these experiments, overnight fasted rats were tube-fed each of the tested tryptophans (5 or 30 mg/100 g body wt) 1 h before killing. Since the results with 5 or 30 mg tryptophan/100 g body weight doses were similar for each group, the results were combined as summarized in Table VI. The results indicate that while the hepatic polyribosomes showed a shift toward heavier aggregation due to control tryptophan, such was not the case following SDA or SDB tryptophan which revealed patterns similar to the control (water-treated) group. In vitro hepatic protein synthesis using microsomal preparations revealed somewhat greater stimulation following control tryptophan than for SDA or SDB tryptophan. In three experiments, we determined hepatic nuclear NTPase activity levels in the control and experimental groups. The means expressed as/zmol Pi released per h per mg protein were as follows: control (water) group, 6.1; TRP (USBC) group, 10.5; SDA group, 10.8; and SDB group, 10.1.

97 TABLE V INHIBITION OF TOTAL IN VITRO [3H]TRYPTOPHAN BINDING USING 2000-FOLD EXCESS OF ~-CARBOLINES Unlabeled test compoundsa

Number of experiments

Hepatic nuclear envelope binding L-TRP

Test compound

Change

(%) ~-Carbolines Norharmane Harmane Harmol Harmine

4 2 2 2

63.6 63.4 73.4 63.4

20.9 22.2 19.4 24.7

-67.1 -65.0 -73.6 -61.0

2 2

73.4 73.4

13.2 23.5

-82.0 -68.0

2

73.4

19.4

-73.6

Harmane- 1,2,3,4-tetrahydro-/~carboline-3-carboxylic acid 2

73.4

20.7

-71.8

69.0

• 9.5

-86.2

Dihydro-~-Carbolines Harmalol Harmaline

Tetrahydro-{$-carbolines Norleagnine (Tetrahydro-/~-carboline)

Compound X ( 1-methyl- 1,2,3,4tetrahydro-/~-carboline3-carboxylic acid) aUnlabeled test compounds were added at 10-4 M.

TABLE VI EFFECT OF TRYPTOPHAN FROM DIFFERENT SOURCES ON HEPATIC POLYRIBOSOMES AND PROTEIN SYNTHESIS 60 min AFTER TUBE-FEEDING a Parameter

Polyribosomal aggregation b In vitro protein synthesisd (% change)

Number of Water determinations

TRP

SDA

6 6

36.1 4- 2.9 +59.5 4. 9.2

42.2 4- 2.2 45.3 4. 2.0 +38.4 4. 13.8 +39.4 ± 12.6

43.2 4. 2.4 c

a Rats were tube-fed 5 or 30 mg tryptophan or water 1 h before killing. b Monomer-dimers/total ribosomes x 100. c Means ± S.E.M.

d Specific activities (counts/rain incorporated into protein/rag RNA).

SDB

98 Discussion

In previous studies, we have investigated whether analogs, metabolites, or related compounds of L-tryptophan as well as other amino acids would elicit a stimulatory response in relation to .hepatic protein synthesis [11,16,21] or compete with [3H]tryptophan binding to hepatic nuclei or nuclear envelopes [10,11] as would Ltryptophan. In general, our results indicated that L-tryptophan alone gave the stimulatory response [8,11,16], yet a number of compounds containing the ot-aminopropionic acid structure did compete to varying degrees with [3H]tryptophan binding to hepatic nuclei or nuclear envelopes [11]. In view of the latter findings which occurred at low concentrations, we considered that the high sensitivity of the in vitro [3H]tryptophan binding assay could possibly detect unknown compounds in relation to the competitive effect on [3H]tryptophan binding to hepatic nuclear envelopes. In this study, our in vitro binding assay was used with batches of Ltryptophan from several.sources, with special attention to one supplier whose Ltryptophan had been implicated in the development of the eosinophilia-myalgia syndrome. Our findings reveal that L-tryptophan obtained from Showa Denko (Lots No. 3206203 and 67116202) contains an impurity, still unknown, which affects in vitro [3H]tryptophan binding to hepatic nuclear envelopes (Table I). Likewise, tube-feeding this L-tryptophan (SDA and SDB) and control L-tryptophan (USBC) to rats and subsequently testing in vitro liver nuclear binding capacity for Ltryptophan demonstrated disparities among the tryptophan products (Table II). The results of the in vitro [3H]tryptophan binding assays with the test compounds (Table I) and those of the in vivo administration of the test compounds followed by in vitro [3H]tryptophan binding assays (Table II) are different (a decrease in specific binding in the former and an increase in specific binding in the latter). Although one can only speculate as to what has happened in vivo, it is conceivable that less nuclear binding occurs in vivo to hepatic nuclear envelopes with SDA and SDB than with control tryptophan (USBC). Thus, when hepatic nuclear envelopes are isolated from rats of each group, more binding sites are available in the experimental than in the control groups and, thus, the in vitro binding assays will show greater increases in specific binding in experimental than in control. Though this explanation may be speculative, testing other biochemical parameters (hepatic polyribosomes and protein synthesis) revealed a modified (somewhat smaller) response with implicated tryptophan than for control tryptophan. In the present study, we observed that tryptophan from one supplier which has been implicated in cases of eosinophilia-myalgia syndrome contains impurity/ies or contaminant/s which affect in vitro [3H]tryptophan binding to hepatic nuclear envelopes. In another report, we have described that certain drugs (benzodiazepines) can influence [3H]tryptophan binding to hepatic nuclear envelopes [22,23]. Specifically, demoxepam, the N-desalkylated compound of chlordiazepoxide, has an inhibitory effect on in vitro [3H]tryptophan binding to rat hepatic nuclei and has an apparent KD -- 22 /~M [22]. In contrast, L-tryptophan has a KD = 18.1 nM [9]. Also, other benzodiazepines, such as chlordiazepoxide, diazepam, prazepam, flurazepam, nordazepam, N-desalkylflurazepam, temazepam, oxazepam, lorazepam, or 4-chlorodiazepam, do not have an appreciable inhibitory effect on in vitro

99

[3H]tryptophan binding to rat hepatic nuclei or nuclear envelopes but when chlordiazepoxide is administered intraperitoneally, the subsequently isolated hepatic nuclei reveal decreased specific tryptophan binding compared to controls [23]. These earlier studies indicate that certain drugs have the ability to affect the capacity of hepatic nuclei to bind to L-tryptophan. Whether these drugs may be acting in a similar manner as the tryptophan impurity/ies of this study remains to be established. Based upon the assay system used in this report, in vitro [3H]tryptophan binding to rat hepatic nuclear envelopes, we have determined that L-tryptophan from Showa Denko, implicated in the eosinophilia-myalgia syndrome, affects the degree of specific binding. This implies that the Showa Denko L-tryptophan contains one or more impurities or contaminants which are responsible for this effect, but it appears not to be the previously implicated 1,1 '-ethylidenebis(tryptophan) or its breakdown product, 1-methyl-l,2,3,4-tetrahydro-/3-carboline-3 carboxylic acid. Thus, our sensitive in vitro assay dealing with a receptor-binding effect has proved to be of value in detecting a biologic difference in the behavior of the implicated Ltryptophan. This may prove of value in searching for the responsible compound/s. At the present time, what the responsible compound/s is/are remains to be determined. We are in the process of evaluating which peak/s obtained by HPLC of the implicated Showa Denko L-tryptophan may be responsible. Also, whether the responsible compound may be involved in inducing the eosinophilia-myalgia syndrome remains highly speculative. References 1 E.A. Beiongia, C.W. Hedberg, G.J. Gleich, K.E. White, A.N. Mayeno, D.A. Loegering, S.L. Dunnette, P.L. Pirie, K.L. MacDonald and M.T. Osterholm, An investigation of the eosinophiliamyalgia syndrome associated with tryptophan use. N. Engl. J. Med., 323 (1990) 357. 2 L. Slutsker, F.C. Hoesly, L. Miller, L.P. Williams, J.C. Watsen and D.W. Fleming, Eosinophiliamyalgia syndrome associated with exposure to tryptophan from a single manufacturer. J. Am. Med. Assoc., 264 (1990) 213. 3 J. Varga, J. Uitto and S.A. Jimenez, The cause and pathogenesis of the eosinophilia-myalgia syndrome. Ann. Int. Med., 116 (1992) 140. 4 Update: Analysis of L-tryptophan for the etiology of eosinophilia-myalgia syndrome. MMWR, 39 (1990) 789. 5 A.N. Mayeno, F. Lin, C.S. Foote, D.A. Locgering, M.M. Ames, C.W. Hedberg and G.J. Gleich, Characterization of "Peak E", a novel amino acid associated with eosinophilia-myalgia syndrome. Science, 250 (1990) 1707. 6 L.J. Crofford, J.I. Rader, M.C. Dalakas, R.H. Hill, Jr., S.W. Page, L.L. Needham, L.S. Brody, M.P. Heyes, R.L. Wilder, P.W. Gold, I.llla, C. Smith and E.M. Sternberg, L-tryptophan implicated in human eosinophilia=myalgia syndrome causes fasciitis and perimyositis in the Lewis rat. J. Clin. Invest., 86 (1990) 1757. 7 L.A. Love, J.I. Rader, S.W. Page, R.H. Hill, R.B. Raybourne and E.M. Sternberg, L-tryptophan and 1,1'=ethylidenebis(tryptophan), a contaminent in eosinophilia-myalgia syndrome caseassociated L-tryptophan, cause myofascial thickening and pancreatic fibrosis in Lewis rats. Arthritis Rheum., 34 (1991) 5131. 8 H. Sidransky, Tryptophan: unique actions by an essential amino acid, in H. Sidransky (Ed.), Nutritional Pathology. Pathobiology of Dietary Imbalances, Marcel Dekker, New York, 1985, p. I. 9 R.N. Kurl, E. Verney and H. Sidransky, Tryptophan-binding sites on nuclear envelopes of rat liver. Nutr. Rep. Inter., 36 (1987) 669.

100 10 H. Sidransky, E. Verney and R. Kurl, Comparison of effects of L-tryptophan and a tryptophan analog, D,L-/~-(1-naphthyl)alanine, on processes relating to hepatic protein synthesis in rats. J. Nutr., 120 (1990) 1157. 11 H. Sidransky, E. Verney, J.W. Cosgrove and A.M. Schwartz, Studies with compounds that compete with tryptophan binding to rat hepatic nuclei. J. Nutr., 122 (1992) 1085. 12 G. Blobel and V.R. Potter, Nuclei from rat liver: isolation method that combines purity with high yield. Science, 154 (1966) 1662. 13 P.S. Agutter and C.D. Gleed, Variability of mammalian liver nuclear envelope preparations. Biochem. J., 192 (1980) 85. 14 J.R. Harris and J.F. Milne, A rapid procedure for the isolation and purification of rat liver nuclear envelopes. Biochem. Soc. Trans., 2 (1974) 1251. 15 R.N. Kurl, E. Verney and H. Sidransky, Identification and immunohistochemical localization of a tryptophan binding protein in nuclear envelopes of rat liver. Arch. Biochem. Biophys., 265 (1988) 286. 16 H. Sidransky, D.S.R. Sarma, M. Bongiorno and E. Verney, Effect of dietary tryptophan on hepatic polyribosomes and protein synthesis in fasted mice. J. Biol. Chem., 243 (1968) 1123. 17 O.H. Lowry, M.R. Rosebrough, A.L. Farr and R.J. Randall, Protein measurement with Folin phenol reagent. J. Biol. Chem., 193 (1951) 265. 18 P.S. Agutter, H.J. McArles and B. McCaldin, Evidence for involvement of nuclear envelope nucleoside triphosphatase in nucleocytoplasmic translocation of ribonucleoprotein. Nature, 263 (1976) 165. 19 G.W. Snedecor and W.G. Cochran, Statistical methods. Iowa State University Press, Ames, IA, 1980, p. 215. 20 J. Ito, Y. Hosaki, Y. Torigoe and K. Sakimoto, Identification of substances formed by decomposition of peak E substance in tryptophan. Food Chem. Toxicol., 30 (1992) 71. 21 H. Sidransky, E. Verney and C.N. Murty, Studies on the influence of tryptophan and related compounds on hepatic polyribosomes and protein synthesis in the rat. J. Nutr., i10 (1980) 2231. 22 H. Sidransky, E. Verney, J.W. Cosgrove and A.M. Schwartz, Inhibitory effect of demoxepam on tryptophan binding to rat hepatic nuclei. Biochem. Med. Met. Biol., 47 (1992) 270. 23 H. Sidransky, E. Verney, J.W. Cosgrove and A.M. Schwartz, Effect of benzodiazepines on tryptophan binding to rat hepatic nuclei. Toxicol. Pathol. (1992) in press.